U.S. patent application number 16/751556 was filed with the patent office on 2020-12-24 for prodrugs of prostate specific membrane antigen (psma) inhibitor.
The applicant listed for this patent is INSTITUTE OF ORGANIC CHEMISTRY AND BIOCHEMISTRY AS CR v.v.i., THE JOHNS HOPKINS UNIVERSITY. Invention is credited to ANDREJ JANCARIK, MARCELA KRECMEROVA, PAVEL MAJER, RANA RAIS, BARBARA SLUSHER, TOMAS TICHY.
Application Number | 20200399298 16/751556 |
Document ID | / |
Family ID | 1000005066202 |
Filed Date | 2020-12-24 |
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United States Patent
Application |
20200399298 |
Kind Code |
A1 |
SLUSHER; BARBARA ; et
al. |
December 24, 2020 |
PRODRUGS OF PROSTATE SPECIFIC MEMBRANE ANTIGEN (PSMA) INHIBITOR
Abstract
Methods and compounds are disclosed for treating a disease or
condition by inhibiting PSMA (Prostate Specific Membrane Antigen)
using prodrugs of 2-PMPA
Inventors: |
SLUSHER; BARBARA;
(KINGSVILLE, MD) ; RAIS; RANA; (KINGSVILLE,
MD) ; KRECMEROVA; MARCELA; (PRAGUE, CZ) ;
TICHY; TOMAS; (PRAGUE, CZ) ; MAJER; PAVEL;
(SYKESVILLE, MD) ; JANCARIK; ANDREJ; (KOPRIVNICE,
CZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY
INSTITUTE OF ORGANIC CHEMISTRY AND BIOCHEMISTRY AS CR
v.v.i. |
Baltimore
Prague |
MD |
US
CZ |
|
|
Family ID: |
1000005066202 |
Appl. No.: |
16/751556 |
Filed: |
January 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
15968074 |
May 1, 2018 |
10544176 |
|
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16751556 |
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15502105 |
Feb 6, 2017 |
9988407 |
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PCT/US15/44053 |
Aug 6, 2015 |
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15968074 |
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62033926 |
Aug 6, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07F 9/65586 20130101;
C07F 9/4084 20130101; C07F 9/4087 20130101; C07F 9/4075 20130101;
C07F 9/4006 20130101; C07F 9/4465 20130101; C07F 9/3808
20130101 |
International
Class: |
C07F 9/6558 20060101
C07F009/6558; C07F 9/44 20060101 C07F009/44; C07F 9/38 20060101
C07F009/38; C07F 9/40 20060101 C07F009/40 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
CA161056 awarded by the National Cancer Institute (NCI). The
government has certain rights in the invention.
Claims
1.-40. (canceled)
41. A composition comprising a dose of a compound of formula (I) or
formula (II) dissolved in an aqueous solution, wherein the aqueous
solution comprises a pharmaceutically acceptable carrier and a
physiologically compatible buffer, and wherein the compound of
formula (I) or formula (II) are: ##STR00148## wherein: (a) each
R.sub.1 is H; each R.sub.2 is selected from the group consisting of
H, alkyl, Ar, --(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; each R.sub.3
is selected from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; and each
R.sub.4 is selected from the group consisting of
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; (b) each
R.sub.1 is alkyl; each R.sub.2 is selected from the group
consisting of H, alkyl, Ar, --(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; each R.sub.3
is selected from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; and each
R.sub.4 is selected from the group consisting of Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O--].sub.m--R.sub-
.7, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; (c) each
R.sub.1 is --(CR.sub.5R.sub.6).sub.n--Ar; each R.sub.2 is selected
from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; each R.sub.3
is selected from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; and each
R.sub.4 is selected from the group consisting of Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; or (d) each
R.sub.1 is selected from Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; each R.sub.2
is selected from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; each R.sub.3
is selected from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; and each
R.sub.4 is selected from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9; wherein:
each n is an integer from 1 to 20; each m is an integer from 1 to
20; each R.sub.5 and R.sub.6 is independently selected from the
group consisting of H, alkyl, and alkylaryl; each R.sub.7 is
independently straight chain or branched alkyl; each Ar is aryl,
substituted aryl, heteroaryl or substituted heteroaryl; each
R.sub.5 and R.sub.9 are independently H or alkyl; and each R.sub.3'
and R.sub.4' are independently H or alkyl; or pharmaceutically
acceptable salts thereof
42. The composition of claim 41, wherein the compound is a compound
of formula (I) and: R.sub.1 is H; R.sub.2 and R.sub.3 are each
selected from the group consisting of H,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7, and
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7; and R.sub.4 is
selected from the group consisting of
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7 and
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7; or
pharmaceutically acceptable salts thereof
43. The composition of claim 41, wherein the compound is a compound
of formula (I) and: R.sub.1 is alkyl; R.sub.2 and R.sub.3 are each
independently selected from the group consisting of H, alkyl,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7 and
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7; and R.sub.4 is
selected from the group consisting of
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7 and
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7; or
pharmaceutically acceptable salts thereof
44. The composition of claim 41, wherein the compound is a compound
of formula (I) and: R.sub.1 is selected from
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7 and
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7; and R.sub.2
R.sub.3, and R.sub.4 are each independently selected from H, Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7, and
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7; or
pharmaceutically acceptable salts thereof
45. The composition of claim 41, wherein the compound is a compound
of formula (I) and: one of R.sub.1, R.sub.2, R.sub.3, or R.sub.4 is
H and the other three are each independently selected from the
group consisting of:
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7 and
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7; wherein
R.sub.5 and R.sub.6 are each independently selected from the group
consisting of H, C.sub.1-8 straight-chain alkyl, and C.sub.1-8
branched-chain alkyl; R.sub.7 is C.sub.1-8 straight-chain alkyl,
and C.sub.1-8 branched-chain alkyl; or pharmaceutically acceptable
salts thereof
46. The composition of claim 41, wherein the compound is a compound
of formula (I) and: R.sub.2 is H; and R.sub.1, R.sub.3, and R.sub.4
are each independently selected from the group consisting of:
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7 and
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7; wherein
R.sub.5 and R.sub.6 are each independently selected from the group
consisting of H, C.sub.1-8 straight-chain alkyl, and C.sub.1-8
branched-chain alkyl; R.sub.7 is C.sub.1-8 straight-chain alkyl or
C.sub.1-8 branched-chain alkyl; or pharmaceutically acceptable
salts thereof
47. The composition of claim 46, wherein R.sub.5 and R.sub.6 are
each H.
48. The composition of claim 41, wherein the compound of formula
(I) is selected from the group consisting of: ##STR00149##
##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154##
##STR00155## or pharmaceutically acceptable salts thereof.
49. The composition of claim 41, wherein the pharmaceutically
acceptable carrier comprises buffered saline.
50. The composition of claim 41, wherein the pharmaceutically
acceptable carrier comprises Hank's solution.
51. The composition of claim 41, wherein the pharmaceutically
acceptable carrier comprises Ringer's solution.
52. The composition of claim 41, wherein the dose of the compound
of formula (I) or formula (II) has a range from about 1 mg to about
50 mg.
53. The composition of claim 41, wherein the dose of the compound
of formula (I) or formula (II) has a range from about 5 mg to about
40 mg.
54. The composition of claim 41, wherein the dose of the compound
of formula (I) or formula (II) has a range from about 10 mg to
about 30 mg.
55. The composition of claim 54, wherein the dose of the compound
of formula (I) or formula (II) is about 10 mg.
56. A method for treating a disease or condition, the method
comprising administering to a subject in need of treatment thereof,
a composition of claim 41 comprising a dose of a compound of
formula (I) or formula (II) dissolved in an aqueous solution,
wherein the aqueous solution comprises a pharmaceutically
acceptable carrier and a physiologically compatible buffer.
57. The method of claim 56, wherein the dose is administered more
than once in a timed-form.
58. The method of claim 56, wherein the dose is administered in
combination with one or more additional therapeutic agents.
59. H The method of claim 56, wherein the disease or condition
comprises a cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/968,074, filed May 1, 2018, which is a
divisional of U.S. patent application Ser. No. 15/502,105, filed
Feb. 6, 2017, now U.S. Pat. No. 9,988,407, which is a .sctn. 371
U.S. National Entry of PCT/US2015/044053, filed Aug. 6, 2015, which
claims the benefit of U.S. Provisional Application No. 62/033,926,
filed Aug. 6, 2014, which is incorporated herein by reference in
its entirety.
BACKGROUND
[0003] The prodrug approach is a well-established strategy to
improve physicochemical, biopharmaceutic and pharmacokinetic
properties of potential drug molecules. Approximately 5-7% of drugs
approved worldwide are prodrugs with annual sales in 2013 of $11.2
billion. Most prodrugs are simple chemical derivatives of the
original molecule. Ester prodrugs, the most common prodrugs,
constitute 49% of all marketed prodrugs. Reasons for the popularity
of ester prodrugs include their generally straight forward
synthesis, their improved lipophilicity and membrane permeability,
and the ubiquitousness of estereases. An example of an approach to
make an ester prodrug is capping the acidic moiety(ies) with
lipophilic alkyl or alkyloxymethyl esters (i.e., pivaloyloxymethyl
(POM) or propyloxycarbonyloxymethyl (POC); e.g., Enalapril,
Adefovir). Another approach is to cap the acidic moiety(ies) with
amino acids to make amides that are recognizable by transporters,
such as Peptide transporter 1 (PEPT1) (e.g., Pomaglumetad
methionil, Valacyclovir).
[0004] PSMA (Prostate Specific Membrane Antigen), also termed GCPII
(glutamate carboxypeptidase II) and FOLH1, is a metallopeptidase
that catalyzes the hydrolysis of N-acetylated aspartate-glutamate
(NAAG) to N-acetyl aspartate (NAA) and glutamate and cleaves
terminal glutamate moieties sequentially from folate polyglutamate
(Ristau et al., 2013; Mesters et al., 2006; Slusher et al., 2013).
One of the most potent, selective, and efficacious PSMA inhibitors
is 2-PMPA (K.sub.i or IC.sub.50=300 .mu.M). After 50-100 mg/kg
intraperitoneal injection (i.p.) doses, it achieves 30-50 .mu.M
concentrations in the brain and provides efficacy in over 20 animal
models of the central nervous system (CNS) or peripheral nervous
system (PNS) including diabetic neuropathy, peripheral neuropathy,
neuropathic pain, general pain, stroke, drug addiction, amyotrophic
lateral sclerosis (ALS), multiple sclerosis (MS), schizophrenia,
epilepsy and several others associated with pathological increase
of glutamate concentration leading to excito-toxic effects and
neuronal death. However, 2-PMPA is a highly polar compound with
multiple carboxylates and a zinc binding group and it has
negligible oral availability. Therefore, in most cases, it must be
dosed intravenously, intraperitoneally, or locally to achieve the
desired effects. This fact limits its potential use as a drug since
most of the above disorders require long term dosing for which the
oral route is strongly preferred.
SUMMARY
[0005] In some aspects, the presently disclosed subject matter
provides a compound of formula (I) or formula (II):
##STR00001##
[0006] wherein:
[0007] each R.sub.1, R.sub.2, R.sub.3, and R.sub.4 is independently
selected from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O].sub.m--R.sub.7-
, --(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9;
[0008] wherein:
[0009] n is an integer from 1 to 20;
[0010] m is an integer from 1 to 20;
[0011] each R.sub.3' and R.sub.4' are independently H or alkyl;
[0012] each R.sub.5 and R.sub.6 is independently selected from the
group consisting of H, alkyl, and alkylaryl;
[0013] each R.sub.7 is independently straightchain or branched
alkyl;
[0014] Ar is aryl, substituted aryl, heteroaryl or substituted
heteroaryl; and
[0015] R.sub.8 and R.sub.9 are each independently H or alkyl;
and
[0016] pharmaceutically acceptable salts thereof.
[0017] In particular aspects, the compound of formula (I) is
selected from the group consisting of:
##STR00002##
[0018] In other aspects, the presently disclosed subject matter
provides a method for treating a disease or a condition, the method
comprising administering to a subject in need of treatment thereof,
a compound of formula (I), a compound of formula (II), or a
pharmaceutical composition thereof, in an amount effective for
treating the disease or condition.
[0019] In particular aspects, the disease or condition is selected
from the group consisting of a neurodegenerative disease, multiple
sclerosis (MS), cancer, angiogenesis, and inflammatory bowel
disease.
[0020] In certain aspects, the neurodegenerative disease is
selected from the group consisting of amyotrophic lateral sclerosis
(ALS), Parkinson's disease (PD), Alzheimer's disease (AD),
Huntington's disease, dementia with Lewy Bodies (DLB),
schizophrenia, pain, epilepsy, stroke, and traumatic brain injury
(TBI).
[0021] In some aspects, the disease or condition results in excess
PSMA activity. In such aspects, the method further comprises
inhibiting the excess PSMA activity when the compound of formula
(I), the compound of formula (II), or a pharmaceutical composition
thereof, is administered.
[0022] Certain aspects of the presently disclosed subject matter
having been stated hereinabove, which are addressed in whole or in
part by the presently disclosed subject matter, other aspects will
become evident as the description proceeds when taken in connection
with the accompanying Examples and Figures as best described herein
below.
BRIEF DESCRIPTION OF THE FIGURES
[0023] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0024] Having thus described the presently disclosed subject matter
in general terms, reference will now be made to the accompanying
Figures, which are not necessarily drawn to scale, and wherein:
[0025] FIG. 1 shows an example of the synthesis of Tris-POC
(JAM0186);
[0026] FIG. 2 shows in vivo bioanalysis via method 1
(2-PMPA+metabolites) and method 2 (2-PMPA selective);
[0027] FIG. 3 shows an embodiment of a representative screening
paradigm;
[0028] FIG. 4 shows an in vitro metabolic stability screen of
compound 1 in human and mouse plasma and liver subcellular
fractions;
[0029] FIG. 5 shows an in vitro metabolic stability screen of
compound 6 in mouse plasma and liver subcellular fractions;
[0030] FIG. 6 shows in vivo single time point pharmacokinetic
studies of compounds 6, 7, and 8 in mice at 30 mg/kg equivalent
2-PMPA showing 30-50 fold enhancement in permeability;
[0031] FIG. 7 shows in vivo pharmacokinetic studies of compounds
JHU 2109, JHU 2110, and JHU 2201 in mice (method 1), showing a
greater then 50-fold increase of POM and POC prodrugs/metabolites
following oral dosing;
[0032] FIG. 8 shows in vivo pharmacokinetic studies of compounds
JHU 2109, JHU 2110, and JHU 2201 (method 2), indicating that POM
and POC ester prodrugs do not release 2-PMPA because the methyl
ester is too stable;
[0033] FIG. 9A and FIG. 9B show: (FIG. 9A) in vitro metabolic
stability screens of compounds JHU 2236 and JHU 2237 in mouse
plasma and liver subcellular fractions (method 1); (FIG. 9B) in
vivo pharmacokinetic studies of compounds JHU 2236, JHU 2237, JHU
2263, JHU 2264, and JHU 2265 (methods 1 (total prodrug exposure)
and 2 (2-PMPA release)), indicating that increasing ester chain
length on carboxylates did not increase 2-PMPA release and no or
minimal 2-PMPA release was observed with ethyl and propyl
ester;
[0034] FIG. 10A and FIG. 10B show: (FIG. 10A) an in vivo single
time point pharmacokinetic study of Tris-POM (compound JAM0168) in
mice, indicating POM esters on carboxylate increases 2-PMPA
approximately 18-fold following oral dosing; and (FIG. 10B) an in
vivo single time point pharmacokinetic study of Tris-POM (compound
JAM0168) in mice (11.69 mg/kg (equiv 2-PMPA); 30 min; N=3);
[0035] FIG. 11A and FIG. 11B show an in vitro metabolic stability
screen of Tris-POC (compound JAM0186) in: (FIG. 11A) human and
mouse plasma and liver subcellular fractions; and (FIG. 11B) human,
dog, and monkey plasma and liver subcellular fractions;
[0036] FIG. 12 shows a single dose pharmacokinetic study in mice
showing plasma 2-PMPA concentrations following 30 mg/kg per oral
administration of Tris-POC (JAM0186; black circles) or 2-PMPA (red
squares) (30 mg/kg (equiv 2-PMPA); 30 min; N=3);
[0037] FIG. 13 shows an in vitro metabolic stability screen of
Tris-POC (JAM0186) in human, dog, and monkey plasma and liver
subcellular fractions;
[0038] FIG. 14 shows a single dose in vivo full time course
pharmacokinetic study of Tris-POC (JAM0186) in dogs (10 mg/kg
(equiv 2-PMPA); N=1) showing a high Cmax;
[0039] FIG. 15 shows an in vitro metabolic stability screen of
Tris-POC (JAM0186) and Tris-methyl-POC in human, dog, and monkey
plasma and liver subcellular fractions;
[0040] FIG. 16 shows a marked increase of PSMA expression in the
villous epithelium from ileal sample of CD patient (Zhang et al.,
2012). Immunohistochemical localization of PSMA (indicated by
arrows) in diseased ileal mucosa from the proximal margin of
resected ileum from an ileal CD subject right panel) and a control
non-IBD subject. Magnification is 100.times.. Bar is 200 mm;
[0041] FIG. 17 shows a marked elevation of PSMA functional
enzymatic activity in the inflamed (diseased) intestinal mucosa of
patients with IBD. PSMA activity was measured from mucosa specimens
(n=32) from diseased (inflamed with active disease) and
normal/uninvolved (macroscopically normal) mucosa from IBD patients
or from non-IBD controls (healthy controls or patients with
diverticulitis]. Note: PSMA is also highly upregulated in colon
cancer;
[0042] FIG. 18 shows that PSMA inhibitor (PSMAi) ameliorates
DNBS-induced colitis as effective as sulfasalazine (Sulfs), an IBD
drug being currently used in the clinic. Mice receiving DNBS to
induce colitis were treated simultaneously with either Sulfs, or
PSMAi (100 mg/kg). Colon weight/body weight ratio, which positively
correlated with the disease activity, was used as a measure for
clinical activity;
[0043] FIG. 19 shows that PSMAi (2-PMPA) also ameliorates disease
activity in DSS-induced murine model of colitis. C57/B6 mice
(approximately 8 weeks old) that were induced to develop colitis
with DSS (2.5%, 7 days in drinking water) were treated
simultaneously with the vehicle or 2-PMPA (100 mg/kg),
respectively. Disease activity index (DAI), which positively
correlated with the disease severity, was used as a measure for
clinical activity. *P<0.05;
[0044] FIG. 20 shows that PSMAi (2-PMPA) effectively suppresses
PSMA activity in the colonic or cecal mucosa of DSS-induced murine
model of colitis. PSMA activity was measured using extract from
mucosa;
[0045] FIG. 21A, FIG. 21B, FIG. 21C and FIG. 21D show that PSMAi
(2-PMPA) treatment leads to not only improvement of disease but
even retraction of prolapse in IL-10 knockout (IL-10KO) mice that
spontaneously develop colitis: (FIG. 21A) improvement of prolapse
and colonic macroscopic disease (inflammation, hypertrophy, stool
inconsistency); (FIG. 21B) body weight after 2-PMPA; (FIG. 21C)
colon weight changes; and (FIG. 21D) prolapse retraction after
treatment. IL-10 KO mice (C57/B6; 3 month old) were treated with
2-PMPA (100 mg/kg) for 2 weeks. *P<0.05;
[0046] FIG. 22 shows a flow-chart of an experimental design to
address whether the PSMA inhibitor directly targets on colonic
epithelial cells (CECs); CACO-2 cell lines or CECs isolated from WT
and IL-10KO mice will be used;
[0047] FIG. 23A and FIG. 23B show the expression of CD103 on human
intestinal DC: (FIG. 23A) FACS dot plot demonstrating
identification of human colonic CD103+ DC from biopsies and
surgical resection tissue obtained from healthy controls or
patients with active CD/UC (inflamed areas), via gating on viable
cells according to forward and side scatter (not shown), HLA-DR
versus lineage cocktail (CD3/CD14/CD16/CD19/CD34), and subsequent
CD103 histogram; and (FIG. 23B) summary graph representing all
experiments (control: n=14; IBD: n=8). T-test was applied
***p<0.001;
[0048] FIG. 24A, FIG. 24B, FIG. 24C and FIG. 24D show the
expression of a437 on murine intestinal DC: (FIG. 24A) FACS dot
plot demonstrating identification of murine colonic CD103+ DC as
CD11c+MHC Class II+ following gating on CD45+ live cells; (FIG.
24B) FACS dot plot demonstrating .alpha..sub.4.beta..sub.7
co-expression with CD103 (markers were co-expressed in all
experiments). Histogram was gated on CD45+ live cells, and
subsequently MHC Class II+CD11c+ cells; (FIG. 24C) FACS histogram
demonstrating example of .alpha..sub.4.beta..sub.7 expression on
murine colonic DC. Histogram was gated on CD45+ live cells, and
subsequently MHC Class II+CD11c+ cells; and (FIG. 24D) summary
graph representing all experiments (n=3 for both). T-test was
applied: *p<0.05;
[0049] FIG. 25 shows PK results with compound JAM0388 after dosing
animals at 30 mg/kg equivalent of 2-PMPA by oral gavage and
collecting brain and plasma samples at 30 min and 5 h. Brain and
plasma samples were quantified for 2-PMPA using our previously
published method (Rais et al, J Pharm Biomed Anal. 2014 January;
88:162-9). JAM0338 demonstrated excellent release of 2-PMPA in
plasma and levels were quantifiable even at 5 h, showing sustained
release of 2-PMPA from the prodrug. Brain levels were low at both
time points;
[0050] FIG. 26 is a schematic illustration demonstrating that GCPII
cleaves NAAG to NAA and glutamate in the brain;
[0051] FIG. 27 demonstrates that GCPII inhibition increases NAAG in
the brain by >30 fold measured by microdialysis;
[0052] FIG. 28A, FIG. 28B and FIG. 28C demonstrate that 2-PMPA
elevates NAAG and improves cognitive function in EAE mice. FIG. 28A
demonstrates that mice show equal cognitive ability in Barnes maze
paths on Day 1. FIG. 28B shows that on Day 4), EAE mice treated
with vehicle show deficit on learning, while EAE mice treated with
2-PMPA have equal cognitive function to healthy control mice. FIG.
28C demonstrates that [NAAG] is elevated in EAE mice treated with
2PMPA; and
[0053] FIG. 29 shows plasma concentration time profiles of 2-PMPA
following i.p. (black line) and oral (red line) administration at
100 mg/kg in mice.
DETAILED DESCRIPTION
[0054] The presently disclosed subject matter now will be described
more fully hereinafter with reference to the accompanying Figures,
in which some, but not all embodiments of the inventions are shown.
Like numbers refer to like elements throughout. The presently
disclosed subject matter may be embodied in many different forms
and should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will satisfy applicable legal requirements. Indeed, many
modifications and other embodiments of the presently disclosed
subject matter set forth herein will come to mind to one skilled in
the art to which the presently disclosed subject matter pertains
having the benefit of the teachings presented in the foregoing
descriptions and the associated Figures. Therefore, it is to be
understood that the presently disclosed subject matter is not to be
limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims.
I. Prodrugs of 2-PMPA
[0055] 2-PMPA is a highly polar compound with multiple carboxylates
and a zinc binding phosphonate group and it has negligible oral
availability. The usual way of solving this problem is by
converting polar groups into less polar functional derivatives.
However, typical prodrug approaches including simple alkyl esters
of the acids such as methyl, ethyl, and propyl were attempted and
were not successful, due to excess stability of these moieties on
the carboxylates. Pivaloyloxymethyl (POM) and
propyloxycarbonyloxymethyl (POC) on phosphonate groups demonstrated
the right combination of lability in vitro and provided the highest
levels of prodrug derived species when dosed orally. Even a
compound with a free .gamma. carboxylate demonstrated good
bioavailability. However, none of these compounds released 2-PMPA
in vivo to any appreciable extent. The presently disclosed subject
matter shows that POM and POC on the bis phosphonate and the alpha
carboxylate were ideal for enhancing the permeability
(approximately 20 fold), as well as release of the parent compound
upon oral dosing.
[0056] Structures of representative structures of 2-PMPA prodrugs
are provided in Table 1. More particularly, the presently disclosed
subject matter includes the capping of the acidic functional groups
of 2-PMPA. In some embodiments, the carboxylic acid groups of
2-PMPA were protected with alkyl esters. See for example, compounds
1, 2, and 3 of Table 1. These carboxylic esters, however,
unexpectedly were too stable in vivo to be effective prodrugs.
Protecting the phosphonate of 2-PMPA with for example bis-POM
(compound 4) or bis-POC (compound 5) while leaving the carboxylates
free, however, was not a feasible solution because of the chemical
instability of such derivatives.
[0057] A combination of both approaches, i.e., protecting the
carboxylic acid groups with an alkyl ester and protecting the
phosphonate with POM or POC, e.g., compounds 6 and 7, provided
compounds that exhibited good permeability. These compounds,
however, were only converted to the corresponding carboxylate
ester, compound 1, which is stable in plasma and did not exhibit
the ability to release 2-PMPA.
[0058] Compounds including POM and POC on the bis-phosphonate of
2-PMPA and an alkyl ester on the .alpha.-carboxylate, with a free
.gamma.-carboxylate, e.g., compounds 8 and 9, exhibited good oral
availability, but were only converted to monoester 10.
[0059] The stability of the simple carboxylic ester, however, can
be overcome by introducing another POC or POM moiety on the
.alpha.-carboxylate (compounds 11 and 12, respectively). Such
compounds exhibited sufficient chemical stability, yet exhibited
the potential to release 2-PMPA.
TABLE-US-00001 TABLE 1 Structures of Representative 2-PMPA Prodrugs
and Metabolic Products IOCB No./ Compound No. Structure MW 2-PMPA
##STR00003## 226.12 1 TT-140113 JHU 2106 ##STR00004## 254.17 2
MK-797 JHU 2236 ##STR00005## 282.23 3 MK-801 JHU 2263 ##STR00006##
310.28 4 ##STR00007## 458.35 5 ##STR00008## 454.41 6 TT-010213 JHU
2110 ##STR00009## 486.41 7 MK-793 JHU 2234 ##STR00010## 472.38 8
TT-150313 JHU 2201 ##STR00011## 468.43 9 ##STR00012## 240.15 10
JAM0186 2-PMPATRIS-POC ##STR00013## 574.47 11 ##STR00014## 582.57
12 TT-041212 JHU 2107 ##STR00015## 791.13 13 TT-250113 JHU 2108
##STR00016## 838.90 14 TT-201212A JHU 2109 ##STR00017## 482.46 15
TT-010213 JHU 2110 ##STR00018## 486.40 16 TT-100113 JHU 2111
##STR00019## 606.58 17 TT-280113 JHU 2112 ##STR00020## 604.60 18
MK798 JHU 2237 ##STR00021## 510.51 19 MK804 JHU 2264 ##STR00022##
482.46 20 MK806 JHU 2265 ##STR00023## 486.40 21 MK-795 JHU 2235
##STR00024## 592.57 22 MK-799 JHU 2238 ##STR00025## 520.68 23
JAM0168 2-PMPA TRIS POM ##STR00026## 568.55 24 JAM0195 JHU 2609
##STR00027## 616.55 25 JAM0191 JHU 2608 ##STR00028## 543.46 26
JAM0196 JHU 2610 ##STR00029## 571.51 27 LTP023 ##STR00030## 522.44
28 LTP120 ##STR00031## 670.60 29 LTP124 ##STR00032## 670.60 30
JAM0388H ##STR00033## 494.43 31 JAM0341H ##STR00034## 540.50 32
TT-120814 ##STR00035## 450.29 33 TT-200714 ##STR00036## 450.29 34
TT-270514 ##STR00037## 540.41 35 TT-011214 ##STR00038## 674.45 36
TT-110814A ##STR00039## 562.37
[0060] In yet other embodiments, fine tuning of the hydrolysis rate
can be evaluated by a combination of POC and methyl-substituted
POC, as illustrated by the following compounds:
##STR00040##
[0061] Further directions in 2-PMPA prodrugs include the following
approach, including more easily hydrolysable phenyl esters;
anhydrides, and dioxolone esters employing paraoxonase for
bioconversion:
##STR00041##
[0062] Additionally, the following dioxolone esters and anhydride
prodrugs of 2-PMPA are contemplated:
##STR00042##
[0063] Further examples of alternative carboxy-esters prodrugs of
2-PMPA also include:
##STR00043##
[0064] Accordingly, in some embodiments, the presently disclosed
subject matter provides a compound of formula (I) or formula
(II):
##STR00044##
[0065] wherein:
[0066] each R.sub.1, R.sub.2, R.sub.3, and R.sub.4 is independently
selected from the group consisting of H, alkyl, Ar,
--(CR.sub.5R.sub.6).sub.n--Ar,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--C(.dbd.O)--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--O--[(CR.sub.5R.sub.6).sub.n--O]m-R.sub.7,
--(CR.sub.5R.sub.6).sub.n--Ar--O--C(.dbd.O)--R.sub.7,
--Ar--C(.dbd.O)--O--(CR.sub.5R.sub.6).sub.n--R.sub.7,
--(CR.sub.5R.sub.6).sub.n--NR.sub.8R.sub.9, and
--(CR.sub.5R.sub.6).sub.n--C(.dbd.O)--NR.sub.8R.sub.9;
[0067] wherein:
[0068] n is an integer from 1 to 20;
[0069] m is an integer from 1 to 20;
[0070] each R.sub.3' and R.sub.4' are independently H or alkyl;
[0071] each R.sub.5 and R.sub.6 is independently selected from the
group consisting of H, alkyl, and alkylaryl;
[0072] each R.sub.7 is independently straightchain or branched
alkyl;
[0073] Ar is aryl, substituted aryl, heteroaryl or substituted
heteroaryl; and
[0074] R.sub.8 and R.sub.9 are each independently H or alkyl;
and
[0075] pharmaceutically acceptable salts thereof.
[0076] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00045##
[0077] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00046##
[0078] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00047##
[0079] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00048##
[0080] In particular embodiments, the compound of formula (I)
is
##STR00049##
[0081] In particular embodiments, the compound of formula (I)
is
##STR00050##
[0082] In particular embodiments, the compound of formula (I)
is
##STR00051##
[0083] In particular embodiments, the compound of formula (I)
is
##STR00052##
[0084] In particular embodiments, the compound of formula (I)
is
##STR00053##
[0085] In particular embodiments, the compound of formula (I)
is
##STR00054##
[0086] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00055##
[0087] In particular embodiments, the compound of formula (I)
is
##STR00056##
[0088] In particular embodiments, the compound of formula (I)
is
##STR00057##
[0089] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00058##
[0090] In particular embodiments, the compound of formula (I)
is
##STR00059##
[0091] In particular embodiments, the compound of formula (I)
is
##STR00060##
[0092] In particular embodiments, the compound of formula (I)
is
##STR00061##
[0093] In particular embodiments, the compound of formula (I)
is
##STR00062##
[0094] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00063##
[0095] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00064##
[0096] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00065##
[0097] In particular embodiments, the compound of formula (I)
is
##STR00066##
[0098] In particular embodiments, the compound of formula (I) is
selected from the group consisting of:
##STR00067##
[0099] In particular embodiments, the compound of formula (II)
is
##STR00068##
[0100] In particular embodiments, the compound of formula (II)
is
##STR00069##
II. Pharmaceutical Compositions and Administration
[0101] In another aspect, the present disclosure provides a
pharmaceutical composition including a compound of formula (I), or
a compound of formula (II), alone or in combination with one or
more additional therapeutic agents in admixture with a
pharmaceutically acceptable excipient. One of skill in the art will
recognize that the pharmaceutical compositions include the
pharmaceutically acceptable salts of the compounds described
above.
[0102] In therapeutic and/or diagnostic applications, the compounds
of the disclosure can be formulated for a variety of modes of
administration, including systemic and topical or localized
administration. Techniques and formulations generally may be found
in
[0103] Remington: The Science and Practice of Pharmacy (20.sup.th
ed.) Lippincott, Williams & Wilkins (2000).
[0104] The compounds according to the disclosure are effective over
a wide dosage range. For example, in the treatment of adult humans,
dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg
per day, and from 5 to 40 mg per day are examples of dosages that
may be used. A non-limiting dosage is 10 to 30 mg per day. The
exact dosage will depend upon the route of administration, the form
in which the compound is administered, the subject to be treated,
the body weight of the subject to be treated, and the preference
and experience of the attending physician.
[0105] Pharmaceutically acceptable salts are generally well known
to those of ordinary skill in the art, and may include, by way of
example but not limitation, acetate, benzenesulfonate, besylate,
benzoate, bicarbonate, bitartrate, bromide, calcium edetate,
carnsylate, carbonate, citrate, edetate, edisylate, estolate,
esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,
lactobionate, malate, maleate, mandelate, mesylate, mucate,
napsylate, nitrate, pamoate (embonate), pantothenate,
phosphate/diphosphate, polygalacturonate, salicylate, stearate,
subacetate, succinate, sulfate, tannate, tartrate, or teoclate.
Other pharmaceutically acceptable salts may be found in, for
example, Remington: The Science and Practice of Pharmacy (20.sup.th
ed.) Lippincott, Williams & Wilkins (2000). Pharmaceutically
acceptable salts include, for example, acetate, benzoate, bromide,
carbonate, citrate, gluconate, hydrobromide, hydrochloride,
maleate, mesylate, napsylate, pamoate (embonate), phosphate,
salicylate, succinate, sulfate, or tartrate.
[0106] Depending on the specific conditions being treated, such
agents may be formulated into liquid or solid dosage forms and
administered systemically or locally. The agents may be delivered,
for example, in a timed- or sustained-low release form as is known
to those skilled in the art. Techniques for formulation and
administration may be found in Remington: The Science and Practice
of Pharmacy (20.sup.th ed.) Lippincott, Williams & Wilkins
(2000). Suitable routes may include oral, buccal, by inhalation
spray, sublingual, rectal, transdermal, vaginal, transmucosal,
nasal or intestinal administration; parenteral delivery, including
intramuscular, subcutaneous, intramedullary injections, as well as
intrathecal, direct intraventricular, intravenous,
intra-articullar, intra-sternal, intra-synovial, intra-hepatic,
intralesional, intracranial, intraperitoneal, intranasal, or
intraocular injections or other modes of delivery.
[0107] For injection, the agents of the disclosure may be
formulated and diluted in aqueous solutions, such as in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological saline buffer. For such
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0108] Use of pharmaceutically acceptable inert carriers to
formulate the compounds herein disclosed for the practice of the
disclosure into dosages suitable for systemic administration is
within the scope of the disclosure. With proper choice of carrier
and suitable manufacturing practice, the compositions of the
present disclosure, in particular, those formulated as solutions,
may be administered parenterally, such as by intravenous injection.
The compounds can be formulated readily using pharmaceutically
acceptable carriers well known in the art into dosages suitable for
oral administration. Such carriers enable the compounds of the
disclosure to be formulated as tablets, pills, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a subject (e.g., patient) to be treated.
[0109] For nasal or inhalation delivery, the agents of the
disclosure also may be formulated by methods known to those of
skill in the art, and may include, for example, but not limited to,
examples of solubilizing, diluting, or dispersing substances such
as, saline, preservatives, such as benzyl alcohol, absorption
promoters, and fluorocarbons.
[0110] Pharmaceutical compositions suitable for use in the present
disclosure include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein.
[0111] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions.
[0112] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipients, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethyl-cellulose (CMC), and/or polyvinylpyrrolidone (PVP:
povidone). If desired, disintegrating agents may be added, such as
the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a
salt thereof such as sodium alginate.
[0113] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol
gel, polyethylene glycol (PEG), and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dye-stuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0114] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin, and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols (PEGs). In
addition, stabilizers may be added.
III. Methods for Treating a Disease or Disorder
[0115] The presently disclosed compounds, which are orally
bioavailable prodrugs of 2-PMPA, allow a clinically acceptable
dosing paradigm for diseases or conditions wherein excess
PSMA/GCPII activity is implicated. These diseases or conditions
include, but are not limited to, neurodegenerative disease such as
amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD),
Alzheimer's disease (AD), Huntington's disease, dementia with Lewy
Bodies (DLB), schizophrenia, pain, epilepsy, stroke, and traumatic
brain injury (TBI), as well as multiple sclerosis (MS), cancer,
angiogenesis and inflammatory bowel disease. As used herein, a
"neurodegenerative disease" is a disease or condition that results
in the progressive loss of the structure and/or function of neurons
in a subject.
[0116] As used herein, the terms "PSMA" or "PSMA polypeptide" refer
to a naturally occurring or endogenous PSMA and to proteins having
an amino acid sequence which is the same as that of a naturally
occurring or endogenous PSMA (e.g., recombinant proteins).
Accordingly, as defined herein, the term includes mature PSMA,
glycosylated or unglycosylated PSMA proteins, polymorphic or
allelic variants, and other isoforms of PSMA (e.g., produced by
alternative splicing or other cellular processes).
[0117] As used herein, an "inhibitor" of PSMA is a molecule that
decreases or inhibits the activity of PSMA when administered. The
inhibitor may interact with PSMA directly or may interact with
another molecule that results in a decrease in the activity of
PSMA.
[0118] The presently disclosed subject matter shows that there is a
marked elevation or excess of PSMA activity in subjects with
certain diseases or conditions. As used herein, the term "excess
PSMA activity" means an increase of PSMA activity in a subject with
a disease or condition as compared to the PSMA activity in a
subject without a similar disease or condition, such as an increase
of approximately 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,
900%, 1000%, or more.
[0119] In some embodiments, the presently disclosed subject matter
provides methods for inhibiting the excess PSMA activity found in a
subject with a disease or condition. As used herein, the term
"inhibit" means to decrease or diminish the excess PSMA activity
found in a subject. The term "inhibit" also may mean to decrease,
suppress, attenuate, diminish, arrest, or stabilize the development
or progression of a disease or condition. Inhibition may occur, for
e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
98%, 99%, or even 100% compared to an untreated control subject or
a subject without the disease or disorder.
[0120] In general, the presently disclosed methods result in a
decrease in the severity of a disease or condition in a subject.
The term "decrease" is meant to inhibit, suppress, attenuate,
diminish, arrest, or stabilize a symptom of a disease or
condition.
[0121] As used herein, the terms "treat," "treating," "treatment,"
and the like refer to reducing or ameliorating a disease or
condition, and/or symptoms associated therewith. It will be
appreciated that, although not precluded, treating a disease or
condition does not require that the disorder, condition or symptoms
associated therewith be completely eliminated.
[0122] Accordingly, in some embodiments, the presently disclosed
subject matter provides a method for treating a disease or a
condition, the method comprising administering to a subject in need
of treatment thereof, a compound of formula (I), a compound of
formula (II), or a pharmaceutical composition thereof, in an amount
effective for treating the disease or condition.
[0123] In particular embodiments, the disease or condition is
selected from the group consisting of a neurodegenerative disease,
multiple sclerosis (MS), cancer, angiogenesis, and inflammatory
bowel disease.
[0124] In certain embodiments, the neurodegenerative disease is
selected from the group consisting of amyotrophic lateral sclerosis
(ALS), Parkinson's disease (PD), Alzheimer's disease (AD),
Huntington's disease, dementia with Lewy Bodies (DLB),
schizophrenia, pain, epilepsy, stroke, and traumatic brain injury
(TBI).
[0125] In some embodiments, the disease or condition results in
excess PSMA activity. In such aspects, the method further comprises
inhibiting the excess PSMA activity when the compound of formula
(I), the compound of formula (II), or a pharmaceutical composition
thereof, is administered.
IV. Definitions
[0126] Although specific terms are employed herein, they are used
in a generic and descriptive sense only and not for purposes of
limitation. Unless otherwise defined, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this presently described
subject matter belongs.
[0127] While the following terms in relation to compounds of
formula (I) are believed to be well understood by one of ordinary
skill in the art, the following definitions are set forth to
facilitate explanation of the presently disclosed subject matter.
These definitions are intended to supplement and illustrate, not
preclude, the definitions that would be apparent to one of ordinary
skill in the art upon review of the present disclosure.
[0128] The terms substituted, whether preceded by the term
"optionally" or not, and substituent, as used herein, refer to the
ability, as appreciated by one skilled in this art, to change one
functional group for another functional group provided that the
valency of all atoms is maintained. When more than one position in
any given structure may be substituted with more than one
substituent selected from a specified group, the substituent may be
either the same or different at every position. The substituents
also may be further substituted (e.g., an aryl group substituent
may have another substituent off it, such as another aryl group,
which is further substituted, for example, with fluorine at one or
more positions).
[0129] Where substituent groups or linking groups are specified by
their conventional chemical formulae, written from left to right,
they equally encompass the chemically identical substituents that
would result from writing the structure from right to left, e.g.,
--CH.sub.2O-- is equivalent to --OCH.sub.2--; --C(.dbd.O)O-- is
equivalent to --OC(.dbd.O)--; --OC(.dbd.O)NR-- is equivalent to
--NRC(.dbd.O)O--, and the like.
[0130] When the term "independently selected" is used, the
substituents being referred to (e.g., R groups, such as groups
R.sub.1, R.sub.2, and the like, or variables, such as "m" and "n"),
can be identical or different. For example, both R.sub.1 and
R.sub.2 can be substituted alkyls, or R.sub.1 can be hydrogen and
R.sub.2 can be a substituted alkyl, and the like.
[0131] The terms "a," "an," or "a(n)," when used in reference to a
group of substituents herein, mean at least one. For example, where
a compound is substituted with "an" alkyl or aryl, the compound is
optionally substituted with at least one alkyl and/or at least one
aryl. Moreover, where a moiety is substituted with an R
substituent, the group may be referred to as "R-substituted." Where
a moiety is R-substituted, the moiety is substituted with at least
one R substituent and each R substituent is optionally
different.
[0132] A named "R" or group will generally have the structure that
is recognized in the art as corresponding to a group having that
name, unless specified otherwise herein. For the purposes of
illustration, certain representative "R" groups as set forth above
are defined below.
[0133] Description of compounds of the present disclosure are
limited by principles of chemical bonding known to those skilled in
the art. Accordingly, where a group may be substituted by one or
more of a number of substituents, such substitutions are selected
so as to comply with principles of chemical bonding and to give
compounds which are not inherently unstable and/or would be known
to one of ordinary skill in the art as likely to be unstable under
ambient conditions, such as aqueous, neutral, and several known
physiological conditions. For example, a heterocycloalkyl or
heteroaryl is attached to the remainder of the molecule via a ring
heteroatom in compliance with principles of chemical bonding known
to those skilled in the art thereby avoiding inherently unstable
compounds.
[0134] The term hydrocarbon, as used herein, refers to any chemical
group comprising hydrogen and carbon. The hydrocarbon may be
substituted or unsubstituted. As would be known to one skilled in
this art, all valencies must be satisfied in making any
substitutions. The hydrocarbon may be unsaturated, saturated,
branched, unbranched, cyclic, polycyclic, or heterocyclic.
Illustrative hydrocarbons are further defined herein below and
include, for example, methyl, ethyl, n-propyl, iso-propyl,
cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl,
cyclohexyl, methoxy, diethylamino, and the like.
[0135] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight (i.e.,
unbranched) or branched chain, acyclic or cyclic hydrocarbon group,
or combination thereof, which may be fully saturated, mono- or
polyunsaturated and can include di- and multivalent groups, having
the number of carbon atoms designated (i.e., C.sub.1-C.sub.10 means
one to ten carbons). In particular embodiments, the term "alkyl"
refers to C.sub.1-20 inclusive, linear (i.e., "straight-chain"),
branched, or cyclic, saturated or at least partially and in some
cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon
radicals derived from a hydrocarbon moiety containing between one
and twenty carbon atoms by removal of a single hydrogen atom.
[0136] Representative saturated hydrocarbon groups include, but are
not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, iso-pentyl,
neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl,
n-undecyl, dodecyl, cyclohexyl, (cyclohexyl)methyl,
cyclopropylmethyl, and homologs and isomers thereof
[0137] "Branched" refers to an alkyl group in which a lower alkyl
group, such as methyl, ethyl or propyl, is attached to a linear
alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to
about 8 carbon atoms (i.e., a C.sub.1-8 alkyl), e.g., 1, 2, 3, 4,
5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group
having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments,
"alkyl" refers, in particular, to C.sub.1-8 straight-chain alkyls.
In other embodiments, "alkyl" refers, in particular, to C.sub.1-8
branched-chain alkyls.
[0138] Alkyl groups can optionally be substituted (a "substituted
alkyl") with one or more alkyl group substituents, which can be the
same or different. The term "alkyl group substituent" includes but
is not limited to alkyl, substituted alkyl, halo, arylamino, acyl,
hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl,
aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There
can be optionally inserted along the alkyl chain one or more
oxygen, sulfur or substituted or unsubstituted nitrogen atoms,
wherein the nitrogen substituent is hydrogen, lower alkyl (also
referred to herein as "alkylaminoalkyl"), or aryl.
[0139] Thus, as used herein, the term "substituted alkyl" includes
alkyl groups, as defined herein, in which one or more atoms or
functional groups of the alkyl group are replaced with another atom
or functional group, including for example, alkyl, substituted
alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro,
amino, alkylamino, dialkylamino, sulfate, and mercapto.
[0140] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon group, or combinations
thereof, consisting of at least one carbon atoms and at least one
heteroatom selected from the group consisting of O, N, P, Si and S,
and wherein the nitrogen, phosphorus, and sulfur atoms may
optionally be oxidized and the nitrogen heteroatom may optionally
be quaternized. The heteroatom(s) O, N, P and S and Si may be
placed at any interior position of the heteroalkyl group or at the
position at which alkyl group is attached to the remainder of the
molecule. Examples include, but are not limited to,
--CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3,
--CH.sub.2--CH.sub.25--S(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3,
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3, O--CH.sub.3,
--O--CH.sub.2--CH.sub.3, and --CN. Up to two or three heteroatoms
may be consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3
and --CH.sub.2--O--Si(CH.sub.3).sub.3.
[0141] As described above, heteroalkyl groups, as used herein,
include those groups that are attached to the remainder of the
molecule through a heteroatom, such as --C(O)R', --C(O)NR',
--NR'R'', --OR', --SR, and/or --SO.sub.2R'. Where "heteroalkyl" is
recited, followed by recitations of specific heteroalkyl groups,
such as --NR'R or the like, it will be understood that the terms
heteroalkyl and --NR'R'' are not redundant or mutually exclusive.
Rather, the specific heteroalkyl groups are recited to add clarity.
Thus, the term "heteroalkyl" should not be interpreted herein as
excluding specific heteroalkyl groups, such as --NR'R'' or the
like.
[0142] "Cyclic" and "cycloalkyl" refer to a non-aromatic mono- or
multicyclic ring system of about 3 to about 10 carbon atoms, e.g.,
3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can
be optionally partially unsaturated. The cycloalkyl group also can
be optionally substituted with an alkyl group substituent as
defined herein, oxo, and/or alkylene. There can be optionally
inserted along the cyclic alkyl chain one or more oxygen, sulfur or
substituted or unsubstituted nitrogen atoms, wherein the nitrogen
substituent is hydrogen, alkyl, substituted alkyl, aryl, or
substituted aryl, thus providing a heterocyclic group.
Representative monocyclic cycloalkyl rings include cyclopentyl,
cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include
adamantyl, octahydronaphthyl, decalin, camphor, camphane, and
noradamantyl, and fused ring systems, such as dihydro- and
tetrahydronaphthalene, and the like.
[0143] The term "cycloalkylalkyl," as used herein, refers to a
cycloalkyl group as defined hereinabove, which is attached to the
parent molecular moiety through an alkyl group, also as defined
above. Examples of cycloalkylalkyl groups include cyclopropylmethyl
and cyclopentylethyl.
[0144] The terms "cycloheteroalkyl" or "heterocycloalkyl" refer to
a non-aromatic ring system, unsaturated or partially unsaturated
ring system, such as a 3- to 10-member substituted or unsubstituted
cycloalkyl ring system, including one or more heteroatoms, which
can be the same or different, and are selected from the group
consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P),
and silicon (Si), and optionally can include one or more double
bonds.
[0145] The cycloheteroalkyl ring can be optionally fused to or
otherwise attached to other cycloheteroalkyl rings and/or
non-aromatic hydrocarbon rings. Heterocyclic rings include those
having from one to three heteroatoms independently selected from
oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur
heteroatoms may optionally be oxidized and the nitrogen heteroatom
may optionally be quaternized. In certain embodiments, the term
heterocylic refers to a non-aromatic 5-, 6-, or 7-membered ring or
a polycyclic group wherein at least one ring atom is a heteroatom
selected from O, S, and N (wherein the nitrogen and sulfur
heteroatoms may be optionally oxidized), including, but not limited
to, a bi- or tri-cyclic group, comprising fused six-membered rings
having between one and three heteroatoms independently selected
from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered
ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2
double bonds, and each 7-membered ring has 0 to 3 double bonds,
(ii) the nitrogen and sulfur heteroatoms may be optionally
oxidized, (iii) the nitrogen heteroatom may optionally be
quaternized, and (iv) any of the above heterocyclic rings may be
fused to an aryl or heteroaryl ring. Representative
cycloheteroalkyl ring systems include, but are not limited to
pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl,
pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl,
quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl,
tetrahydrofuranyl, and the like.
[0146] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like. The terms "cycloalkylene" and
"heterocycloalkylene" refer to the divalent derivatives of
cycloalkyl and heterocycloalkyl, respectively.
[0147] An unsaturated alkyl group is one having one or more double
bonds or triple bonds. Examples of unsaturated alkyl groups
include, but are not limited to, vinyl, 2-propenyl, crotyl,
2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,
3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the
higher homologs and isomers. Alkyl groups which are limited to
hydrocarbon groups are termed "homoalkyl."
[0148] More particularly, the term "alkenyl" as used herein refers
to a monovalent group derived from a C.sub.1-20 inclusive straight
or branched hydrocarbon moiety having at least one carbon-carbon
double bond by the removal of a single hydrogen atom. Alkenyl
groups include, for example, ethenyl (i.e., vinyl), propenyl,
butenyl, 1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl, and
butadienyl.
[0149] The term "cycloalkenyl" as used herein refers to a cyclic
hydrocarbon containing at least one carbon-carbon double bond.
Examples of cycloalkenyl groups include cyclopropenyl,
cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl,
1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and
cyclooctenyl.
[0150] The term "alkynyl" as used herein refers to a monovalent
group derived from a straight or branched C.sub.1-20 hydrocarbon of
a designed number of carbon atoms containing at least one
carbon-carbon triple bond. Examples of "alkynyl" include ethynyl,
2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, heptynyl,
and allenyl groups, and the like.
[0151] The term "alkylene" by itself or a part of another
substituent refers to a straight or branched bivalent aliphatic
hydrocarbon group derived from an alkyl group having from 1 to
about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group
can be straight, branched or cyclic. The alkylene group also can be
optionally unsaturated and/or substituted with one or more "alkyl
group substituents." There can be optionally inserted along the
alkylene group one or more oxygen, sulfur or substituted or
unsubstituted nitrogen atoms (also referred to herein as
"alkylaminoalkyl"), wherein the nitrogen substituent is alkyl as
previously described. Exemplary alkylene groups include methylene
(--CH.sub.2--); ethylene (--CH.sub.2--CH.sub.2--); propylene
(--(CH.sub.2).sub.3--); cyclohexylene (--C.sub.6H.sub.10);
--CH.dbd.CH--CH.dbd.CH--CH; CH.dbd.CH--CH.sub.2--;
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.dbd.CHCH.sub.2--, --CH.sub.2CsCCH.sub.2--,
--CH.sub.2CH.sub.2CH(CH.sub.2CH.sub.2CH.sub.3)CH.sub.2--,
--(CH.sub.2).sub.q--N(R)--(CH.sub.2).sub.r--, wherein each of q and
r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20,
and R is hydrogen or lower alkyl; methylenedioxyl
(--O--CH.sub.2--O--); and ethylenedioxyl (--O--
(CH.sub.2).sub.2-O--). An alkylene group can have about 2 to about
3 carbon atoms and can further have 6-20 carbons. Typically, an
alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with
those groups having 10 or fewer carbon atoms being some embodiments
of the present disclosure. A "lower alkyl" or "lower alkylene" is a
shorter chain alkyl or alkylene group, generally having eight or
fewer carbon atoms.
[0152] The term "heteroalkylene" by itself or as part of another
substituent means a divalent group derived from heteroalkyl, as
exemplified, but not limited by,
--CH.sub.2--CH.sub.2-S--CH.sub.2--CH.sub.2-and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxo, alkylenedioxo,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula --C(O)OR'--
represents both --C(O)OR'-- and --R'OC(O)--.
[0153] The term "aryl" means, unless otherwise stated, an aromatic
hydrocarbon substituent that can be a single ring or multiple rings
(such as from 1 to 3 rings), which are fused together or linked
covalently. The term "heteroaryl" refers to aryl groups (or rings)
that contain from one to four heteroatoms (in each separate ring in
the case of multiple rings) selected from N, O, and S, wherein the
nitrogen and sulfur atoms are optionally oxidized, and the nitrogen
atom(s) are optionally quaternized. A heteroaryl group can be
attached to the remainder of the molecule through a carbon or
heteroatom. Non-limiting examples of aryl and heteroaryl groups
include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,
pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,
3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,
5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,
3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,
purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below. The terms "arylene" and "heteroarylene" refer to
the divalent forms of aryl and heteroaryl, respectively.
[0154] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxo, arylthioxo, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the terms
"arylalkyl" and "heteroarylalkyl" are meant to include those groups
in which an aryl or heteroaryl group is attached to an alkyl group
(e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like)
including those alkyl groups in which a carbon atom (e.g., a
methylene group) has been replaced by, for example, an oxygen atom
(e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,
and the like). However, the term "haloaryl," as used herein is
meant to cover only aryls substituted with one or more
halogens.
[0155] Where a heteroalkyl, heterocycloalkyl, or heteroaryl
includes a specific number of members (e.g. "3 to 7 membered"), the
term "member" refers to a carbon or heteroatom.
[0156] Further, a structure represented generally by the
formula:
##STR00070##
as used herein refers to a ring structure, for example, but not
limited to a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a
7-carbon, and the like, aliphatic and/or aromatic cyclic compound,
including a saturated ring structure, a partially saturated ring
structure, and an unsaturated ring structure, comprising a
substituent R group, wherein the R group can be present or absent,
and when present, one or more R groups can each be substituted on
one or more available carbon atoms of the ring structure. The
presence or absence of the R group and number of R groups is
determined by the value of the variable "n," which is an integer
generally having a value ranging from 0 to the number of carbon
atoms on the ring available for substitution. Each R group, if more
than one, is substituted on an available carbon of the ring
structure rather than on another R group. For example, the
structure above where n is 0 to 2 would comprise compound groups
including, but not limited to:
##STR00071##
and the like.
[0157] A dashed line representing a bond in a cyclic ring structure
indicates that the bond can be either present or absent in the
ring. That is, a dashed line representing a bond in a cyclic ring
structure indicates that the ring structure is selected from the
group consisting of a saturated ring structure, a partially
saturated ring structure, and an unsaturated ring structure.
[0158] The symbol () denotes the point of attachment of a moiety to
the remainder of the molecule.
[0159] When a named atom of an aromatic ring or a heterocyclic
aromatic ring is defined as being "absent," the named atom is
replaced by a direct bond.
[0160] Each of above terms (e.g., "alkyl," "heteroalkyl,"
"cycloalkyl, and "heterocycloalkyl", "aryl," "heteroaryl,"
"phosphonate," and "sulfonate" as well as their divalent
derivatives) are meant to include both substituted and
unsubstituted forms of the indicated group. Optional substituents
for each type of group are provided below.
[0161] Substituents for alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl monovalent and divalent derivative groups
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to:
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --C(O)NR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R'", --NR''C(O)OR',
--NR--C(NR'R").dbd.NR'", --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R", --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such groups. R', R'', R''' and R'''' each may
independently refer to hydrogen, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl (e.g., aryl substituted with 1-3 halogens), substituted or
unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl
groups. As used herein, an "alkoxy" group is an alkyl attached to
the remainder of the molecule through a divalent oxygen. When a
compound of the disclosure includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 4-, 5-, 6-,
or 7-membered ring. For example, --NR'R'' is meant to include, but
not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0162] Similar to the substituents described for alkyl groups
above, exemplary substituents for aryl and heteroaryl groups (as
well as their divalent derivatives) are varied and are selected
from, for example: halogen, --OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --C(O)NR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''', --NR''C(O)OR',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR'''--S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and
--NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxo, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
aromatic ring system; and where R', R'', R''' and R'''' may be
independently selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl and substituted
or unsubstituted heteroaryl. When a compound of the disclosure
includes more than one R group, for example, each of the R groups
is independently selected as are each R', R'', R''' and R''''
groups when more than one of these groups is present.
[0163] Two of the substituents on adjacent atoms of aryl or
heteroaryl ring may optionally form a ring of the formula
-T-C(O)--(CRR').sub.q--U--, wherein T and U are independently
--NR--, --O--, --CRR'-- or a single bond, and q is an integer of
from 0 to 3. Alternatively, two of the substituents on adjacent
atoms of aryl or heteroaryl ring may optionally be replaced with a
substituent of the formula -A-(CH.sub.2).sub.r--B--, wherein A and
B are independently --CRR'--, --O--, --NR--, --S--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2NR'-- or a single bond, and r is an
integer of from 1 to 4.
[0164] One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula
--(CRR').sub.s--X'--(C''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X' is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R'' may be independently selected from
hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl.
[0165] As used herein, the term "acyl" refers to an organic acid
group wherein the --OH of the carboxyl group has been replaced with
another substituent and has the general formula RC(.dbd.O)--,
wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic,
heterocyclic, or aromatic heterocyclic group as defined herein). As
such, the term "acyl" specifically includes arylacyl groups, such
as an acetylfuran and a phenacyl group. Specific examples of acyl
groups include acetyl and benzoyl.
[0166] The terms "alkoxyl" or "alkoxy" are used interchangeably
herein and refer to a saturated (i.e., alkyl-O--) or unsaturated
(i.e., alkenyl-O--and alkynyl-O--) group attached to the parent
molecular moiety through an oxygen atom, wherein the terms "alkyl,"
"alkenyl," and "alkynyl" are as previously described and can
include C1-20 inclusive, linear, branched, or cyclic, saturated or
unsaturated oxo-hydrocarbon chains, including, for example,
methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl,
t-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the
like.
[0167] The term "alkoxyalkyl" as used herein refers to an
alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl
group.
[0168] "Aryloxyl" refers to an aryl-O-- group wherein the aryl
group is as previously described, including a substituted aryl. The
term "aryloxyl" as used herein can refer to phenyloxyl or
hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl
substituted phenyloxyl or hexyloxyl.
[0169] "Aralkyl" refers to an aryl-alkyl-group wherein aryl and
alkyl are as previously described, and included substituted aryl
and substituted alkyl. Exemplary aralkyl groups include benzyl,
phenylethyl, and naphthylmethyl.
[0170] "Aralkyloxyl" refers to an aralkyl-O-- group wherein the
aralkyl group is as previously described. An exemplary aralkyloxyl
group is benzyloxyl.
[0171] "Alkoxycarbonyl" refers to an alkyl-O-CO-- group. Exemplary
alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,
butyloxycarbonyl, and t-butyloxycarbonyl.
[0172] "Aryloxycarbonyl" refers to an aryl-O-CO-- group. Exemplary
aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
[0173] "Aralkoxycarbonyl" refers to an aralkyl-O-CO-- group. An
exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
[0174] "Carbamoyl" refers to an amide group of the formula
--CONH.sub.2. "Alkylcarbamoyl" refers to a R'RN--CO-- group wherein
one of R and R' is hydrogen and the other of R and R' is alkyl
and/or substituted alkyl as previously described.
"Dialkylcarbamoyl" refers to a R'RN--CO-- group wherein each of R
and R' is independently alkyl and/or substituted alkyl as
previously described.
[0175] The term carbonyldioxyl, as used herein, refers to a
carbonate group of the formula --O--CO--OR.
[0176] "Acyloxyl" refers to an acyl-O-- group wherein acyl is as
previously described.
[0177] The term "amino" refers to the --NH.sub.2 group and also
refers to a nitrogen containing group as is known in the art
derived from ammonia by the replacement of one or more hydrogen
radicals by organic radicals. For example, the terms "acylamino"
and "alkylamino" refer to specific N-substituted organic radicals
with acyl and alkyl substituent groups respectively.
[0178] An "aminoalkyl" as used herein refers to an amino group
covalently bound to an alkylene linker. More particularly, the
terms alkylamino, dialkylamino, and trialkylamino as used herein
refer to one, two, or three, respectively, alkyl groups, as
previously defined, attached to the parent molecular moiety through
a nitrogen atom. The term alkylamino refers to a group having the
structure --NHR' wherein R' is an alkyl group, as previously
defined; whereas the term dialkylamino refers to a group having the
structure --NR'R'', wherein R' and R'' are each independently
selected from the group consisting of alkyl groups. The term
trialkylamino refers to a group having the structure --NR'R''R''',
wherein R', R'', and R'' are each independently selected from the
group consisting of alkyl groups. Additionally, R', R'', and/or R''
taken together may optionally be --(CH.sub.2).sub.k-- where k is an
integer from 2 to 6. Examples include, but are not limited to,
methylamino, dimethylamino, ethylamino, diethylamino,
diethylaminocarbonyl, methylethylamino, iso-propylamino,
piperidino, trimethylamino, and propylamino.
[0179] The amino group is --NR'R'', wherein R' and R'' are
typically selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl.
[0180] The terms alkylthioether and thioalkoxyl refer to a
saturated (i.e., alkyl-S--) or unsaturated (i.e., alkenyl-S-- and
alkynyl-S--) group attached to the parent molecular moiety through
a sulfur atom. Examples of thioalkoxyl moieties include, but are
not limited to, methylthio, ethylthio, propylthio, isopropylthio,
n-butylthio, and the like.
[0181] "Acylamino" refers to an acyl-NH-- group wherein acyl is as
previously described. "Aroylamino" refers to an aroyl-NH-- group
wherein aroyl is as previously described.
[0182] The term "carbonyl" refers to the --(C.dbd.O)-- group.
[0183] The term "carboxyl" refers to the --COOH group. Such groups
also are referred to herein as a "carboxylic acid" moiety.
[0184] The terms "halo," "halide," or "halogen" as used herein
refer to fluoro, chloro, bromo, and iodo groups. Additionally,
terms such as "haloalkyl," are meant to include monohaloalkyl and
polyhaloalkyl. For example, the term "halo(C.sub.1-C4)alkyl" is
mean to include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0185] The term "hydroxyl" refers to the --OH group.
[0186] The term "hydroxyalkyl" refers to an alkyl group substituted
with an --OH group.
[0187] The term "mercapto" refers to the --SH group.
[0188] The term "oxo" as used herein means an oxygen atom that is
double bonded to a carbon atom or to another element.
[0189] The term "nitro" refers to the --NO.sub.2 group.
[0190] The term "thio" refers to a compound described previously
herein wherein a carbon or oxygen atom is replaced by a sulfur
atom.
[0191] The term "sulfate" refers to the --SO.sub.4 group.
[0192] The term thiohydroxyl or thiol, as used herein, refers to
--SH.
[0193] The term ureido refers to a urea group of the formula
--NH--CO--NH.sub.2.
[0194] Unless otherwise explicitly defined, a "substituent group,"
as used herein, includes a functional group selected from one or
more of the following moieties, which are defined herein:
[0195] (A) --OH, --NH.sub.2, --SH, --CN, --CF.sub.3, --NO.sub.2,
oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,
unsubstituted aryl, unsubstituted heteroaryl, and
[0196] (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
and heteroaryl, substituted with at least one substituent selected
from:
[0197] (i) oxo, --OH, --NH.sub.2, --SH, --CN, --CF.sub.3,
--NO.sub.2, halogen, unsubstituted alkyl, unsubstituted
heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,
and
[0198] (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
and heteroaryl, substituted with at least one substituent selected
from:
[0199] (a) oxo, --OH, --NH.sub.2, --SH, --CN, --CF.sub.3,
--NO.sub.2, halogen, unsubstituted alkyl, unsubstituted
heteroalkyl, unsubstituted cycloalkyl, unsubstituted
heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,
and
[0200] (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,
or heteroaryl, substituted with at least one substituent selected
from oxo, --OH, --NH.sub.2, --SH, --CN, --CF.sub.3, --NO.sub.2,
halogen, unsubstituted alkyl, unsubstituted heteroalkyl,
unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,
unsubstituted aryl, and unsubstituted heteroaryl.
[0201] A "lower substituent" or "lower substituent group," as used
herein means a group selected from all of the substituents
described hereinabove for a "substituent group," wherein each
substituted or unsubstituted alkyl is a substituted or
unsubstituted C.sub.1-C.sub.8 alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8
membered heteroalkyl, each substituted or unsubstituted cycloalkyl
is a substituted or unsubstituted C.sub.5-C.sub.7 cycloalkyl, and
each substituted or unsubstituted heterocycloalkyl is a substituted
or unsubstituted 5 to 7 membered heterocycloalkyl.
[0202] A "size-limited substituent" or "size-limited substituent
group," as used herein means a group selected from all of the
substituents described above for a "substituent group," wherein
each substituted or unsubstituted alkyl is a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20
membered heteroalkyl, each substituted or unsubstituted cycloalkyl
is a substituted or unsubstituted C.sub.4-C.sub.8 cycloalkyl, and
each substituted or unsubstituted heterocycloalkyl is a substituted
or unsubstituted 4 to 8 membered heterocycloalkyl.
[0203] Throughout the specification and claims, a given chemical
formula or name shall encompass all tautomers, congeners, and
optical- and stereoisomers, as well as racemic mixtures where such
isomers and mixtures exist.
[0204] Certain compounds of the present disclosure possess
asymmetric carbon atoms (optical or chiral centers) or double
bonds; the enantiomers, racemates, diastereomers, tautomers,
geometric isomers, stereoisometric forms that may be defined, in
terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or
(L)-for amino acids, and individual isomers are encompassed within
the scope of the present disclosure. The compounds of the present
disclosure do not include those which are known in art to be too
unstable to synthesize and/or isolate. The present disclosure is
meant to include compounds in racemic and optically pure forms.
Optically active (R)- and (S)-, or (D)- and (L)-isomers may be
prepared using chiral synthons or chiral reagents, or resolved
using conventional techniques. When the compounds described herein
contain olefenic bonds or other centers of geometric asymmetry, and
unless specified otherwise, it is intended that the compounds
include both E and Z geometric isomers.
[0205] Unless otherwise stated, structures depicted herein are also
meant to include all stereochemical forms of the structure; i.e.,
the R and S configurations for each asymmetric center. Therefore,
single stereochemical isomers as well as enantiomeric and
diastereomeric mixtures of the present compounds are within the
scope of the disclosure.
[0206] It will be apparent to one skilled in the art that certain
compounds of this disclosure may exist in tautomeric forms, all
such tautomeric forms of the compounds being within the scope of
the disclosure. The term "tautomer," as used herein, refers to one
of two or more structural isomers which exist in equilibrium and
which are readily converted from one isomeric form to another.
[0207] Unless otherwise stated, structures depicted herein are also
meant to include compounds which differ only in the presence of one
or more isotopically enriched atoms. For example, compounds having
the present structures except for the replacement of a hydrogen by
a deuterium or tritium, or the replacement of a carbon by .sup.13C-
or .sup.14C-enriched carbon are within the scope of this
disclosure.
[0208] The compounds of the present disclosure may also contain
unnatural proportions of atomic isotopes at one or more of atoms
that constitute such compounds. For example, the compounds may be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). All
isotopic variations of the compounds of the present disclosure,
whether radioactive or not, are encompassed within the scope of the
present disclosure.
[0209] As used herein the term "monomer" refers to a molecule that
can undergo polymerization, thereby contributing constitutional
units to the essential structure of a macromolecule or polymer.
[0210] A "polymer" is a molecule of high relative molecule mass,
the structure of which essentially comprises the multiple
repetition of unit derived from molecules of low relative molecular
mass, i.e., a monomer.
[0211] As used herein, an "oligomer" includes a few monomer units,
for example, in contrast to a polymer that potentially can comprise
an unlimited number of monomers. Dimers, trimers, and tetramers are
non-limiting examples of oligomers.
[0212] The compounds of the present disclosure may exist as salts.
The present disclosure includes such salts. Examples of applicable
salt forms include hydrochlorides, hydrobromides, sulfates,
methanesulfonates, nitrates, maleates, acetates, citrates,
fumarates, tartrates (e.g. (+)-tartrates, (-)-tartrates or mixtures
thereof including racemic mixtures, succinates, benzoates and salts
with amino acids such as glutamic acid. These salts may be prepared
by methods known to those skilled in art. Also included are base
addition salts such as sodium, potassium, calcium, ammonium,
organic amino, or magnesium salt, or a similar salt. When compounds
of the present disclosure contain relatively basic functionalities,
acid addition salts can be obtained by contacting the neutral form
of such compounds with a sufficient amount of the desired acid,
either neat or in a suitable inert solvent. Examples of acceptable
acid addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived organic acids like acetic, propionic,
isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,
lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,
citric, tartaric, methanesulfonic, and the like. Also included are
salts of amino acids such as arginate and the like, and salts of
organic acids like glucuronic or galactunoric acids and the like.
Certain specific compounds of the present disclosure contain both
basic and acidic functionalities that allow the compounds to be
converted into either base or acid addition salts.
[0213] The neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in the conventional manner. The parent form of the
compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents.
[0214] Certain compounds of the present disclosure can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are encompassed within the scope of the present
disclosure. Certain compounds of the present disclosure may exist
in multiple crystalline or amorphous forms. In general, all
physical forms are equivalent for the uses contemplated by the
present disclosure and are intended to be within the scope of the
present disclosure.
[0215] The term "pharmaceutically acceptable salts" is meant to
include salts of active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituent moieties found on the compounds described herein. When
compounds of the present disclosure contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amino, or magnesium salt, or
a similar salt. When compounds of the present disclosure contain
relatively basic functionalities, acid addition salts can be
obtained by contacting the neutral form of such compounds with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of pharmaceutically acceptable acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like {see, for
example, Berge et al, "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds
of the present disclosure contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0216] In addition to salt forms, the present disclosure provides
compounds, which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present disclosure. Additionally, prodrugs can be converted to
the compounds of the present disclosure by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present disclosure when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0217] The term "protecting group" refers to chemical moieties that
block some or all reactive moieties of a compound and prevent such
moieties from participating in chemical reactions until the
protective group is removed, for example, those moieties listed and
described in T. W. Greene, P. G. M. Wuts, Protective Groups in
Organic Synthesis, 3rd ed. John Wiley & Sons (1999). It may be
advantageous, where different protecting groups are employed, that
each (different) protective group be removable by a different
means. Protective groups that are cleaved under totally disparate
reaction conditions allow differential removal of such protecting
groups. For example, protective groups can be removed by acid,
base, and hydrogenolysis. Groups such as trityl, dimethoxytrityl,
acetal and tert-butyldimethylsilyl are acid labile and may be used
to protect carboxy and hydroxy reactive moieties in the presence of
amino groups protected with Cbz groups, which are removable by
hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic
acid and hydroxy reactive moieties may be blocked with base labile
groups such as, without limitation, methyl, ethyl, and acetyl in
the presence of amines blocked with acid labile groups such as
tert-butyl carbamate or with carbamates that are both acid and base
stable but hydrolytically removable.
[0218] Carboxylic acid and hydroxy reactive moieties may also be
blocked with hydrolytically removable protective groups such as the
benzyl group, while amine groups capable of hydrogen bonding with
acids may be blocked with base labile groups such as Fmoc.
Carboxylic acid reactive moieties may be blocked with
oxidatively-removable protective groups such as
2,4-dimethoxybenzyl, while co-existing amino groups may be blocked
with fluoride labile silyl carbamates.
[0219] Allyl blocking groups are useful in the presence of acid-
and base-protecting groups since the former are stable and can be
subsequently removed by metal or pi-acid catalysts. For example, an
allyl-blocked carboxylic acid can be deprotected with a
palladium(O)-catalyzed reaction in the presence of acid labile
t-butyl carbamate or base-labile acetate amine protecting groups.
Yet another form of protecting group is a resin to which a compound
or intermediate may be attached. As long as the residue is attached
to the resin, that functional group is blocked and cannot react.
Once released from the resin, the functional group is available to
react.
[0220] Typical blocking/protecting groups include, but are not
limited to the following moieties:
##STR00072##
[0221] The subject treated by the presently disclosed methods in
their many embodiments is desirably a human subject, although it is
to be understood that the methods described herein are effective
with respect to all vertebrate species, which are intended to be
included in the term "subject." Accordingly, a "subject" can
include a human subject for medical purposes, such as for the
treatment of an existing condition or disease or the prophylactic
treatment for preventing the onset of a condition or disease, or an
animal subject for medical, veterinary purposes, or developmental
purposes. Suitable animal subjects include mammals including, but
not limited to, primates, e.g., humans, monkeys, apes, and the
like; bovines, e.g., cattle, oxen, and the like; ovines, e.g.,
sheep and the like; caprines, e.g., goats and the like; porcines,
e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys,
zebras, and the like; felines, including wild and domestic cats;
canines, including dogs; lagomorphs, including rabbits, hares, and
the like; and rodents, including mice, rats, and the like. An
animal may be a transgenic animal. In some embodiments, the subject
is a human including, but not limited to, fetal, neonatal, infant,
juvenile, and adult subjects. Further, a "subject" can include a
patient afflicted with or suspected of being afflicted with a
condition or disease. Thus, the terms "subject" and "patient" are
used interchangeably herein.
[0222] In general, the "effective amount" of an active agent or
drug delivery device refers to the amount necessary to elicit the
desired biological response. As will be appreciated by those of
ordinary skill in this art, the effective amount of an agent or
device may vary depending on such factors as the desired biological
endpoint, the agent to be delivered, the composition of the
encapsulating matrix, the target tissue, and the like.
[0223] Following long-standing patent law convention, the terms
"a," "an," and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a subject" includes a plurality of subjects, unless the context
clearly is to the contrary (e.g., a plurality of subjects), and so
forth.
[0224] Throughout this specification and the claims, the terms
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires otherwise.
Likewise, the term "include" and its grammatical variants are
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that can be
substituted or added to the listed items.
[0225] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing amounts, sizes,
dimensions, proportions, shapes, formulations, parameters,
percentages, parameters, quantities, characteristics, and other
numerical values used in the specification and claims, are to be
understood as being modified in all instances by the term "about"
even though the term "about" may not expressly appear with the
value, amount or range. Accordingly, unless indicated to the
contrary, the numerical parameters set forth in the following
specification and attached claims are not and need not be exact,
but may be approximate and/or larger or smaller as desired,
reflecting tolerances, conversion factors, rounding off,
measurement error and the like, and other factors known to those of
skill in the art depending on the desired properties sought to be
obtained by the presently disclosed subject matter. For example,
the term "about," when referring to a value can be meant to
encompass variations of, in some embodiments, .+-.100% in some
embodiments .+-.50%, in some embodiments .+-.20%, in some
embodiments .+-.10%, in some embodiments .+-.5%, in some
embodiments .+-.1%, in some embodiments .+-.0.5%, and in some
embodiments .+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed methods or employ the
disclosed compositions.
[0226] Further, the term "about" when used in connection with one
or more numbers or numerical ranges, should be understood to refer
to all such numbers, including all numbers in a range and modifies
that range by extending the boundaries above and below the
numerical values set forth. The recitation of numerical ranges by
endpoints includes all numbers, e.g., whole integers, including
fractions thereof, subsumed within that range (for example, the
recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as
fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and
any range within that range.
EXAMPLES
[0227] The following Examples have been included to provide
guidance to one of ordinary skill in the art for practicing
representative embodiments of the presently disclosed subject
matter. In light of the present disclosure and the general level of
skill in the art, those of skill can appreciate that the following
Examples are intended to be exemplary only and that numerous
changes, modifications, and alterations can be employed without
departing from the scope of the presently disclosed subject matter.
The synthetic descriptions and specific examples that follow are
only intended for the purposes of illustration, and are not to be
construed as limiting in any manner to make compounds of the
disclosure by other methods.
Example 1
Methods
[0228] In Vitro Stability Studies:
[0229] The stock solution for most prodrugs was prepared as a 10 mM
solution in DMSO except JHU 2107, which was solubilized in THF
(tetrahydrofuran) to carry out the in vitro studies.
[0230] The chemical stability of prodrugs was evaluated using
simulated gastric fluid (pH 1.2) and Hanks' Balanced Salt Solution
(HBSS) buffer (pH 7.4). Briefly, prodrugs were spiked (10 .mu.M) in
respective solutions and incubated at 37.degree. C. for 1 h. At
predetermined time points (0, 30 and 60 min), aliquots of 1004 were
removed and diluted with 1004 of water. Prodrug disappearance was
monitored using the developed liquid chromatography and tandem mass
spectrometry (LC/MS/MS) method described below.
[0231] For metabolic stability, plasma (mouse, dog, monkey and
human) and liver microsomes (mouse, dog, monkey and human) were
used. For stability, prodrugs (10 .mu.M) were spiked in each matrix
and incubated in an orbital shaker at 37.degree. C. At
predetermined times (0, 30 and 60 min), 100 .mu.L aliquots of the
mixture in triplicate were removed and the reaction quenched by
addition of three times the volume of ice cold acetonitrile spiked
with the internal standard (losartan 504). The samples were
vortexed for 30 s and centrifuged 12000 g for 10 min. 50 .mu.L
supernatant diluted with 50 .mu.L water was transferred to a 250
.mu.L polypropylene vial sealed with a Teflon cap. Prodrug
disappearance was monitored over time using a liquid chromatography
and tandem mass spectrometry (LC/MS/MS) method as described
below.
[0232] For LC/MS/MS, prodrugs were separated with Thermo Scientific
Accela UPLC system coupled to Accela open autosampler on an Agilent
C18 (100.times.2.1 mm id) UPLC column. The autosampler was
temperature controlled and operating at 10.degree. C. The mobile
phase used for the chromatographic separation was composed of
acetonitrile/water containing 0.1% formic acid and was run at a
flow rate of 0.5 mL/minute for 4.5 minutes using gradient elution.
The column effluent was monitored using TSQ Vantage
triple-quadrupole mass spectrometric detector, equipped with an
electrospray probe set in the positive ionization mode. Samples
were introduced into the ionization source through a heated
nebulized probe (350.degree. C.).
[0233] For quantification of compound remaining, disappearance of
prodrugs was measured from ratio of peak areas of analyte to IS.
Percentage remaining was calculated in the following manner:
Avg . Response * 60 min Avg . Response 0 min .times. 100
##EQU00001## where response=[(Area of analyte)/(Area of internal
standard)]
*Average response is average of two samples at each time point.
[0234] In Vivo Pharmacokinetics of 2-PMPA Prodrugs in Rodent (Mice)
and Non-Rodent (Dogs) Species:
[0235] Prodrugs were dosed peroral (30 mg/kg equiv. 2-PMPA) in mice
at a dosing volume of 1 mL/kg. Blood was obtained via cardiac
puncture and tissue dissected at 0 min, 15 min, 30 min, 1 h, 2 h,
and 4 h post dose (n=3 per time point). Single time point studies
were conducted at 30 min (N=3) following dosing. Plasma was
harvested from blood by centrifugation. Mean concentration-time
data was used for pharmacokinetic (PK) analysis.
Non-compartmental-analysis module in WinNonlin.RTM. (version 5.3)
was used to assess pharmacokinetic parameters. Peak plasma
concentrations (C.sub.max) and time to C.sub.max (T.sub.max) were
the observed values. Area under the curve (AUC) was calculated by
log-linear (p.o.) trapezoidal rule to the end of sample collection
(AUClast) and extrapolated to infinity (AUG.sub.0-.infin..) by
dividing the last quantifiable concentration by the terminal
disposition rate constant (k.sub.e). Terminal half-life (t.sub.1/2)
was estimated from first order kinetics: t.sub.1/2=0.693/k.sub.e.
The goal was to find prodrugs yielding oral bioavailability %
F.gtoreq.30%.
[0236] For pharmacokinetics in beagle dogs, animals were dosed with
2-PMPA prodrug (10 mg/kg equivalent 2-PMPA) p.o. (by mouth). Blood
samples were collected from the jugular vein (.about.1 mL) via
direct venipuncture, placed into potassium oxalate with sodium
fluoride tubes, and maintained on wet ice until processed. Blood
samples were centrifuged at a temperature of 4.degree. C., at
3000.times.g, for 5 minutes. Blood samples were maintained chilled
throughout processing. Plasma was collected in tubes and flash
frozen. Samples were stored in a freezer set to maintain
-60.degree. C. to -80.degree. C. until further analysis.
[0237] Bioanalysis of 2-PMPA Prodrugs in Plasma and Tissue:
[0238] 2-PMPA concentrations in plasma and tissue samples were
determined using two different methods (FIG. 2). Method 1 showed
the total amount of the prodrug in the sample following oral dosing
(a measure of the disappearance of the prodrug) and method 2
evaluated the specific release of 2-PMPA in plasma and tissues from
the prodrug. In vitro screening showed metabolic instability of
almost all the prodrugs tested.
[0239] Method 1 involved use of a strong derivatizing reagent,
n-butanol with 3N HCl, which converted the prodrug and its
metabolites including 2-PMPA into a 2-PMPA butyl ester to obtain
the total exposures from the prodrug. Briefly, prior to extraction,
frozen samples were thawed on ice. For plasma extraction, 50 .mu.L
of the calibration standards or samples were transferred into
silanized microcentrifuge tubes. Sample preparation involved a
single liquid extraction by addition of 300 .mu.L of methanol as
extraction solution with internal standard (i.e., 5 .mu.M of 2-PMSA
in methanol), followed by vortexing for 30 s and then
centrifugation at 12000 g for 10 min. Supernatant was transferred
(.about.250 .mu.L) and evaporated to dryness at 40.degree. C. under
a gentle stream of nitrogen. The residue was reconstituted with 100
.mu.L of derivatizing agent, n-butanol with 3N HCl, and samples
were vortexed. The samples were heated at -60.degree. C. in a
shaking water bath for 30 min. At the end of 30 min, the
derivatized samples were allowed to cool at room temperature and
dried again for removal of derivatizing reagent, under a gentle
stream of nitrogen. The residue was reconstituted in 100 .mu.L of
30% acetonitrile in water v/v. The samples were vortexed and
centrifuged again. 804 supernatant was transferred to a 250 .mu.L
polypropylene vial sealed with a Teflon cap and a volume of 10
.mu.L was injected onto the ultra-performance liquid chromatography
(UPLC) instrument for quantitative analysis. Chromatographic
analysis was performed using an Accela.TM. ultra high-performance
system consisting of an analytical pump, and an autosampler coupled
with TSQ Vantage mass spectrometer (Thermo Fisher Scientific Inc.,
Waltham Mass.). Separation of the analyte from potentially
interfering material was achieved at ambient temperature using
Agilent Eclipse Plus column (100.times.2.1 mm i.d.) packed with a
1.8 .mu.m C18 stationary phase. The mobile phase used was composed
of 0.1% formic acid in acetonitrile and 0.1% formic acid in
H.sub.2O with gradient elution, starting with 20% (organic)
linearly increasing to 65% up to 2.5 min, maintaining at 65%
(2.5-3.5 min) and reequilibrating to 30% by 5 min. The total run
time for each analyte was 5.0 min. The [M+H].sup.+ ion transitions
of derivatized 2-PMPA at m/z 325.522>121.296, 195.345 and that
of the internal standard at m/z 339.537>191.354, 149.308, were
monitored.
[0240] Method 2 was a gentle method to evaluate specific release of
2-PMPA in plasma and tissues from prodrug. Briefly, 2-PMPA was
extracted from plasma by protein precipitation with 5.times.
methanol containing 2-(phosphonomethyl) succinic acid (2-PMSA; 104)
as an internal standard. For brain tissue extraction, the samples
were weighed in a 1.7 mL silanized tubes to which 4 times the
volume of methanol (dilution 1:5) was added. The tissues were
stored in -20.degree. C. for 1 h and then homogenized. The
calibration curve for the tissues was developed using naive mouse
brains from untreated animals as a matrix. For sciatic nerve, the
nerves were weighed and homogenized in 504, methanol and the
calibration curve was developed using naive sciatic nerves from
untreated animals as a matrix. The samples were vortexed and
centrifuged. For tissue extraction, either 50 .mu.L (brain) or 254
(sciatic nerve) of the calibration standards or samples were
transferred into silanized microcentrifuge tubes. Sample
preparation involved a single liquid extraction by addition of 150
.mu.L of methanol as extraction solution with internal standard
(i.e., 5 .mu.M of 2-PMSA in methanol). Supernatant was dried under
a gentle stream of nitrogen at 45.degree. C. and the residue
reconstituted with 75 .mu.L of acetonitrile and vortexed. 25 .mu.L
of derivatizing agent
N-tert-Butyldimethysilyl-N-methyltrifluoro-acetamide (MTBSTFA) was
added to microcentrifuge tubes, vortexed, and heated at
.about.60.degree. C. for 40 min. At the end of 40 min, the
derivatized samples .about.75 .mu.L were transferred to a 250 .mu.L
polypropylene vials and were analyzed via LC/MS/MS. Chromatographic
analysis was performed using an Accela.TM. ultra high-performance
system consisting of an analytical pump, and an autosampler coupled
with TSQ Vantage mass spectrometer (Thermo Fisher Scientific Inc.,
Waltham Mass.). Separation of the analyte from potentially
interfering material was achieved at ambient temperature using
Waters X-terra.RTM., RP18, 3.5 .mu.m, and (2.1.times.50 mm). The
mobile phase used was composed of 0.1% formic acid in acetonitrile
and 0.1% formic acid in H.sub.2O with gradient elution, starting
with 90% (organic) linearly increasing to 99% up to 2.5 min,
maintaining at 99% (2.5-4.0 min) and reequilibrating to 90% by 5
min. The total run time for each analyte was 5.0 min.
Chromatographic analysis will be performed on Accela UPLC. The
[M+H].sup.+ ion transitions of derivatized 2-PMPA at m/z
683.0>551.4 and that of the internal standard at m/z
669.0>537.2 were monitored with the total run time of 5 min.
Avg . Response * 60 min Avg . Response 0 min .times. 100
##EQU00002## Where,Response=[(Area of analyte)/(Area of internal
standard)]
*Average response is average of two samples at each time point.
Example 2
Compound Preparation
[0241] General Procedures:
[0242] The .sup.1H NMR spectra were measured at 400.13. .sup.1H NMR
spectra are standardized to the internal signal of TMS (.delta.
0.0, CDCl.sub.3). The chemical shifts are given in .delta.-scale,
the coupling constants J are given in Hz. The IR spectra were
measured in CHCl.sub.3 on FT-IR spectrometer Bruker Equinox 55. Low
and high resolution CI mass spectra were measured using an
orthogonal acceleration time-of-flight (OA-TOF) mass spectrometer
(GCT premier, Waters) at an ionising voltage of 70 eV, the m/z
values are given with their relative intensities (%). The spectra
were recorded in positive mode and the source temperature was
150.degree. C. Methane was present as a reagent gas in the CI
source. For exact measurement the spectra were internally
calibrated using Heptacosa or
2,4,6-tris(trifluoromethyl)-1,3,5-triazine (Metri). The ESI mass
spectra were recorded with a ZQ micromass mass spectrometer
(Waters) equipped with an ESCi multi-mode ion source and controlled
by MassLynx software. THF was freshly distilled from
sodium/benzophenone under nitrogen. The flash chromatography was
performed on Silica gel 60 (0.040-0.063 mm, Fluka).
##STR00073##
JAM0105
##STR00074##
[0244] This compound was prepared from known literature. .sup.1H
NMR and .sup.13C NMR spectra were in agreement with the published
data.
JAM0106
##STR00075##
[0246] The same method of preparation as for previous compound
JAM0131.
[0247] Compound JAM0105 (6.62 g, 21.62 mmol),
N,N-Dimethylmethyleneiminium iodide (10 g, 54.05 mmol, 2.5 equiv.)
Absolute methanol (265 mL). Reaction mixture was stirred at
65.degree. C. h. The organic solvent was evaporated in vacuo. The
residue was filtered through pad of silica gel (hexane-ethyl
acetate 5:1) to afford the desired product (5.02 g, 94%) as an oil.
1H NMR and 13C NMR spectra were in agreement with the published
data.
JAM0113
##STR00076##
[0249] The same method of preparation as for previous compound
JAM0106. Compound JAM0105 (6.62 g, 21.62 mmol),
N,N-Dimethylmethyleneiminium iodide (10 g, 54.05 mmol, 2.5 equiv.)
Absolute ethanol (265 mL). Reaction mixture was stirred at
78.degree. C. overnight. The organic solvent was evaporated in
vacuo. The residue was filtered through pad of silica gel
(hexane-ethyl acetate 10:1 to 5:1) to afford the desired product
(5.25 g, 93%) as an oil. 1H NMR and 13C NMR spectra were in
agreement with the published data.
JAM0131
##STR00077##
[0251] A dry Schlenk flask was charged with the previous compound
JAM0105 (6.62 g, 21.62 mmol), N,N-Dimethylmethyleneiminium iodide
(10 g, 54.05 mmol, 2.5 equiv.) and then it was flushed with argon.
Absolute t-BuOH (265 mL) was added to the flask and the mixture was
stirred at 65.degree. C. for 48 h. The organic solvent was
evaporated in vacuo. The residue was filtered through pad of silica
gel (hexane-ethyl acetate 5: 1) to afford the desired product (5 g,
80%) as an oil.
[0252] ESI MS: 313 (M+Na.sup.+).
[0253] HR ESI MS: calcd for C.sub.17H.sub.22O.sub.4Na 313.14103;
found 313.14106.
JAM0109
##STR00078##
[0255] The same method of preparation as for previous compound
JAM0149. Diethyl phosphite (2.6 mL, 20.14 mmol), A solution of
trimethylaluminium (2 M in hexanes, 10 mL, 20.14 mmol, 1.0 equiv.)
JAM0106 (5 g, 20.14 mmol, 1.0 equiv.) dichloromethane (70 mL).
Filtration through pad of silica gel (hexane-ethyl acetate 1:1 to
3:1) Product (7.3 g, 94%) as an oil.
[0256] ESI MS: 323 (M+Na.sup.+). HR ESI MS: calcd for
C.sub.17H.sub.35NO.sub.2SNa 340.22807; found 340.22805.
JAM0114
##STR00079##
[0258] The same method of preparation as for previous compound
JAM0149. Diethyl phosphite (2.58 mL, 20 mmol), A solution of
trimethylaluminium (2 M in hexanes, 10 mL, 20 mmol, 1.0 equiv.)
JAM0113 (5.25 g, 20 mmol, 1.0 equiv.) dichloromethane (70 mL).
Filtration through pad of silica gel (hexane-ethyl acetate 1:1 to
3:1) Product (7.5 g, 94%) as an oil.
[0259] ESI MS: 323 (M+Na.sup.+).
[0260] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0149
##STR00080##
[0262] Diethyl phosphite (8.5 mL, 66.1 mmol, 1 equiv.) was
dissolved in absolute dichloromethane (57 mL) under argon and
cooled to 0.degree. C. A solution of trimethyl aluminium (2 M in
hexanes, 33 mL, 66.1 mmol, 1 equiv.) was added dropwise and the
solution was stirred at 0.degree. C. for 30 min. Solution of the
compound JAM0131 (19.2 g, 66.1 mmol, 1 equiv) in dichloromethane
(171 mL) was added and the cooling bath was removed. The reaction
mixture was then stirred at room temperature overnight. The
reaction was quenched with 2 N hydrochloric acid (40 mL). Then it
was extracted with diethyl ether (3.times.40 mL), the combined
organic layers were washed with water (40 mL), brine (40 mL), and
dried over anhydrous MgSO4. The evaporation of the solvents
afforded an oil, which was filtered through pad of silica gel
(hexane-ethyl acetate 3: 1 to 1: 1) to afford the desired product
(28.3 g, 94%) as an oil.
[0263] ESI MS: 323 (M+Na.sup.+).
[0264] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
##STR00081##
JAM0 112
##STR00082##
[0266] A Schlank flask was charged with Ethyl acrylate (15 mL, 0.14
mol) under argon, then TDAP (5 mL, 28 mmol, 20 mol %) was slowly
added and the reactin mixture was stirred at 60.degree. C. for 2 h.
Product was destillated of on Kugelrohr aparatus (100.degree. C. at
0.1 mbar). 1H NMR and 13C NMR spectra were in agreement with the
published data.
JAM0115
##STR00083##
[0268] A Schlank flask was charged with ethyl acrylate (15 mL, 0.12
mol) under argon, then TDAP (4.4 mL, 24 mmol, 20 mol %) was slowly
added and the reactin mixture was stirred at 60.degree. C. for 2 h.
Product was destillated of on Kugelrohr apparatus (125.degree. C.
at 0.1 mbar).
JAM0116
##STR00084##
[0270] The same method of preparation as for previous compound
JAM0149. Diethyl phosphite (4.5 mL, 35 mmol), A solution of
trimethylaluminium (2 M in hexanes, 17.5 mL, 35 mmol, 1.0 equiv.)
JAM0112 (7.0 g, 35 mmol, 1.0 equiv.) dichloromethane (120 mL).
Filtration through pad of silica gel (hexane-ethyl acetate 1:1 to
3:1) Product JAM0116 (11 g, 94%) as an oil.
[0271] 1H NMR and 13C NMR spectra were in agreement with the
published data.
JAM0117
##STR00085##
[0273] The same method of preparation as for previous compound
JAM0149. Diethyl phosphite (2.7 mL, 21 mmol), A solution of
trimethylaluminium (2 M in hexanes, 10.5 mL, 21 mmol, 1.0 equiv.)
JAM0115 (4.8 g, 21 mmol, 1.0 equiv.) dichloromethane (70 mL).
Filtration through pad of silica gel (hexane-ethyl acetate 1:1 to
3:1) Product JAM0117 (7.2 g, 94%) as an oil. .sup.1H NMR (400 MHz,
CDCl.sub.3): 0.92 (3H, t, J=7.4), 0.94 (3H, t, J=7.4), 1.28-1.32
(6H, m), 1.59-1.70 (4H, m), 1.79-1.89 (1H, m), 1.92-2.05 (2H, m),
2.19-2.40 (3H, m), 2.74-2.84 (1H, m), 4.00-4.12 (8H, m).
[0274] .sup.31P NMR (162 MHz, CDCl.sub.3): 28.55
[0275] ESI MS: 389 (M+Na.sup.+).
[0276] HR ESI MS: calcd for C.sub.16H.sub.31O.sub.2NaP 389.16996;
found 389.16869.
##STR00086##
JAM0151
##STR00087##
[0278] Phosphonate JAM0149 (25.4 g, 60 mmol), was dissolved in
dichloromethane (100 mL) and trifluoroacetic acid (100 mL) was
slowly added. The reaction mixture was stirred at room temperature
overnight. Then the solvents were removed in vacuo. The residue was
filtered through a short pad of silica gel (chloroform-methanol 10:
1) to furnish the desired product (19.7 g, 88%) as an oil.
[0279] ESI MS: 395 (M+Na.sup.+).
[0280] HR ESI MS: calcd for C.sub.17H.sub.35O.sub.7NaP 395.12301;
found 395.12337.
JAM0153
##STR00088##
[0282] Dry flask was charged with phosphonate JAM0151 (1.95 g, 5.24
mmol), NaI (1.57 g, 7.86 mmol, 2 equiv.), triethylamine (1.1 mL,
7.86 mmol, 1.5 equiv.). Dry DMF was added and reaction mixture was
stirred at room temperature for 15 min. and then Chloromethyl
pivalate (1.5 mL, 10.47 mmol, 2 equiv.) was slowly added. Reaction
mixture was stirred at room temperature overnight. The solvent was
removed under reduced pressure and the residue was chromatographed
on silica gel (ethyl acetate-hexane 40: 1) to afford the desired
product (1.38 g, 54%) as an oil.
[0283] ESI MS: 323 (M+Na.sup.+).
[0284] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0162
##STR00089##
[0286] The same method of preparation as for previous compound
JAM0153. Phosphonate JAM0151 (2.15 g, 5.77 mmol), NaI (1.73 g,
11.55 mmol, 2 equiv.), triethylamine (1.21 mL, 8.66 mmol, 1.5
equiv.), Chloromethyl isopropyl carbonate (1.55 mL, 11.55 mmol, 2
equiv.), DMF (30 mL). Chromatography on silica gel (hexane-ethyl
acetate 2:1). Product (1.97 g, 70%) as an oil.
[0287] ESI MS: 323 (M+Na.sup.+).
[0288] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0182
##STR00090##
[0290] The same method of preparation as for previous compound
JAM0153. Phosphonate JAM0151 (2 g, 5.37 mmol), NaI (1.61 g, 10.74
mmol, 2 equiv.), triethylamine (1.5 mL, 10.74 mmol, 2 equiv.),
1-Chloroethyl isopropyl carbonate (1.64 mL, 10.74 mmol, 2 equiv.),
DMF (30 mL). Chromatography on silica gel (hexane-ethyl acetate
1:1). Product (1.1 g, 21%) as an oil.
[0291] ESI MS: 323 (M+Na.sup.+).
[0292] HR ESI MS: calcd for C.sub.17F.sub.135NO.sub.2SNa 340.22807;
found 340.22805.
JAM0179
##STR00091##
[0294] The same method of preparation as for previous compound
JAM0153. Phosphonate JAM0151 (1.0 g, 2.68 mmol), NaI (805 mg, 5.37
mmol, 2 equiv.), triethylamine (750 .mu.L, 5.37 mmol, 2 equiv.),
2-Chloro-N,N-dimethylacetamide (552 .mu.L, 5.37 mmol, 2 equiv.),
DMF (14 mL). Chromatography on silica gel (chloroform-methanol
20:1). Product (1 g, 85%) as an oil.
[0295] ESI MS: 323 (M+Na.sup.+).
[0296] HR ESI MS: calcd for C.sub.17F.sub.135NO.sub.2SNa 340.22807;
found 340.22805.
JAM0154
##STR00092##
[0298] The compound JAM0153 (3.3 g, 6.78 mmol) was dissolved in
absolute dichloromethane (40 mL) under argon and cooled to
0.degree. C. A bromotrimethylsilane (3.6 mL, 27.13 mmol, 4 equiv.)
was added dropwise and the solution was stirred at 0.degree. C.
overnight. The volatiles were removed in vacuo and the residue was
diluted with mixture of methanol and toluene (3.times.30 mL, 1: 1)
and evaporated to obtain desired product (2.77 mg, 95%) as an oil
and directly used in next reaction without characterization.
JAM0164
##STR00093##
[0300] The same method of preparation as for previous compound
JAM0154.Phosphonate JAM0162 (1.6 g, 3.28 mmol), TMSBr (1.54 mL,
11.68 mmol, 4 equiv.), DCM (20 mL). Product (1.39 g, 98%) obtained
as an oil.
JAM0185
##STR00094##
[0302] The same method of preparation as for previous compound
JAM0154. Phosphonate JAM0182 (770 mg, 1.53 mmol), TMSBr (809 .mu.L,
6.13 mmol, 4 equiv.), DCM (10 mL). Product (669 mg, 98%) obtained
as an oil.
JAM0184
##STR00095##
[0304] The same method of preparation as for previous compound
JAM0154. Phosphonate JAM0179 (1.05 g, 2.30 mmol), TMSBr (1.21 mL,
9.18 mmol, 4 equiv.), DCM (13 mL). Product (904 mg, 98%) obtained
as an oil.
##STR00096##
JAM0167
##STR00097##
[0306] Dry Schlank flask was charged with previous compound JAM0154
(600 mg, 1.39 mmol) and dissolved in dry dioxane (7 mL). DBU (0.42
mL, 2.80 mmol, 2 equiv), Chloromethyl pivalate (0.8 mL, 5.58 mmol,
4 equiv.) was added and the reaction mixture was stirred at
100.degree. C. for 6 h. The volatiles were removed in vacuo and the
residue was chromatographed on silica gel (toluene-aceton 10:1 to
afford impure desired product (48 mg, 5.2%) as an oil. The product
was further purified using preparative scale HPLC (gradient 10:50,
Rt=12.5 min.).
[0307] ESI MS: 323 (M+Na.sup.+).
[0308] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0166
##STR00098##
[0310] The same method of preparation as for previous compound
JAM0167. Phosphonate JAM0164 (1.14 g, 2.64 mmol), DBU (0.79 mL,
5.28 mmol, 2 equiv), Chloromethyl isopropyl carbonate (3.5 mL,
26.40 mmol, 10 equiv.), dioxane (14 mL). Chromatography on silica
gel (toluene-aceton 5:1). Product (mg, <10%) as an oil. The
product was further purified using preparative scale HPLC (gradient
10:50, Rt=12.5 min.).
[0311] ESI MS: 323 (M+Na.sup.+).
[0312] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0189
##STR00099##
[0314] Dry Schlank flask was charged with previous compound JAM0185
(574 mg, 1.29 mmol), K.sub.2CO.sub.3 (550 mg, 3.98 mmol, 3.1
equiv.) and dissolved in dry DMF (12 mL). 1-Chloroethyl isopropyl
carbonate (2 mL, 12.86 mmol, 10 equiv.) was added and the reaction
mixture was stirred at 60.degree. C. for 6 h. The volatiles were
removed in vacuo and the residue was chromatographed on silica gel
(toluene-aceton 7:1) to afford impure desired product (88 mg, 10%)
as an oil. The product was further purified using preparative scale
HPLC (gradient 10:50, Rt=12.5 min.).
[0315] ESI MS: 323 (M+Na.sup.+).
[0316] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0188
##STR00100##
[0318] The same method of preparation as for previous compound
JAM0167. Phosphonate JAM0184 (555 mg, 1.38 mmol), DBU (433 .mu.L,
2.9 mmol, 2.1 equiv), Chloromethyl isopropyl carbonate (1.85 mL,
13.82 mmol, 10 equiv.), dioxane (7 mL). Chromatography on silica
gel (chloroform-methanol 40:1). Product (96 mg, 11%) as an oil. The
product was further purified using preparative scale HPLC (gradient
10:50, Rt=12.5 min.).
[0319] ESI MS: 656 (M+Na.sup.+).
[0320] HR ESI MS: calcd for C.sub.27H.sub.40O.sub.14NNaP 656.20786;
found 656.20772
JAM0187
##STR00101##
[0322] The same method of preparation as for previous compound
JAM0189. Phosphonate JAM0184 (200 mg, 0.498 mmol), K.sub.2CO.sub.3
(344 mg, 2.49 mmol, 5 equiv.), 1-Chloroethyl isopropyl carbonate
(304 .mu.L, 1.99 mmol, 2 equiv.), DMF (6 mL). Chromatography on
silica gel (chloroform-methanol 10:1). Product (92 mg, 28%) as an
oil. The product was further purified using preparative scale HPLC
(gradient 10:50, Rt=12.5 min.).
[0323] ESI MS: 323 (M+Na.sup.+).
[0324] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805
JAM0168
##STR00102##
[0326] The previous compound JAM0167 (48 mg, 72.9 mmol) was
dissolved in dry THF (3 mL). 10% Palladium on carbon (5 mg) was
added and reaction mixture was bubbled with hydrogen for 10 min.
Reaction mixture was stirred at room temperature overnight under
hydrogen atmosphere. Palladium was filtered through cotton and the
volatiles were removed in vacuo to afford desired product (40 mg,
98%) as an oil.
[0327] ESI MS: 323 (M+Na.sup.+).
[0328] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0186
##STR00103##
[0330] The same method of preparation as for previous compound
JAM0168 (FIG. 1). Phosphonate (1.18 g, 1.77 mmol), 10% palladium on
carbon (100 mg), THF (70 mL). Product (996 mg, 98%) as an oil.
[0331] ESI MS: 323 (M+Na.sup.+).
[0332] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0195
##STR00104##
[0334] The same method of preparation as for previous compound
JAM0168. Phosphonate JAM0189 (1.0 g, 1.42 mmol), 10% palladium on
carbon (100 mg), THF (45 mL). Product (587 mg, 98%) as an oil.
[0335] ESI MS: 323 (M+Na.sup.+).
[0336] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805.
JAM0191
##STR00105##
[0338] The same method of preparation as for previous compound
JAM0168. Phosphonate JAM0188 (100 mg, 0.158 mmol), 10% palladium on
carbon (10 mg), THF (5 mL). Product (84 mg, 98%) as an oil.
[0339] ESI MS: 566 (M+Na.sup.+).
[0340] HR ESI MS: calcd for C.sub.20H.sub.34O.sub.14NNaP 566.16091;
found 566.16087
JAM0196
##STR00106##
[0342] The same method of preparation as for previous compound
JAM0168. Phosphonate JAM0187 (100 mg, 0.151 mmol), 10% palladium on
carbon (10 mg), THF (5 mL). Product (84 mg, 98%) as an oil.
[0343] ESI MS: 594 (M+Na.sup.+).
[0344] HR ESI MS: calcd for C.sub.22H.sub.38O.sub.14NaP 594.19221;
found 594.19215
JAM0214
##STR00107##
[0346] A flask was charged with phosphonate JAM0151 (2.0 g, 5.37
mmol), DCC (1.22 g, 5.91 mmol, 1.1 equiv.), DMAP (65.6 mg, 0.54
mmol, 10 mol %). Dry dichloromethane was added and the reaction
mixture was stirred at room temperature for 15 min. Then
4-Acetamidophenol (974 mg, 6.44 mmol, 1.2 equiv.) was added in one
portion. Reaction mixture was stirred at room temperature
overnight. N,N-Dicyclohexylurea was filtered off and the organic
solvent was evaporated in vacuo. The residue was chromatographed on
silica gel (chloroform-methanol 20: 1) to afford the desired
product (1.55 g, 57%) as an oil.
[0347] ESI MS: 528 (M+Na.sup.+).
[0348] HR ESI MS: calcd for C.sub.25H.sub.32O.sub.8NNaP 528.17577;
found 528.17598
JAM0216
##STR00108##
[0350] The same method of preparation as for previous compound
JAM0154. Phosphonate JAM0214 (1.24 g, 2.48 mmol), TMSBr (1.31 mL,
9.92 mmol, 4 equiv.), DCM (16 mL). Product (1.1 g, 98%) obtained as
an oil.
JAM0218
##STR00109##
[0352] The same method of preparation as for previous compound
JAM0189. Phosphonate JAM0216 (200 mg, 0.498 mmol), K.sub.2CO.sub.3
(344 mg, 2.49 mmol, 5 equiv.), 1-Chloroethyl isopropyl carbonate
(304 .mu.L, 1.99 mmol, 2 equiv.), DMF (6 mL). Chromatography on
silica gel (chloroform-methanol 10:1). Product (90 mg, 25%) as an
oil. The product was further purified using preparative scale HPLC
(gradient 10:50, Rt=12.5 min.).
[0353] ESI MS: 323 (M+Na.sup.+).
[0354] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805
JAM0219
##STR00110##
[0356] The same method of preparation as for previous compound
JAM0168. Phosphonate JAM0218 (100 mg, 0.158 mmol), 10% palladium on
carbon (10 mg), THF (5 mL). Product JAM0218 (92 mg, 98%) as an
oil.
[0357] ESI MS: 323 (M+Na.sup.+).
[0358] HR ESI MS: calcd for C.sub.17H.sub.35NO.sub.2SNa 340.22807;
found 340.22805
[0359] .sup.1H NMR (400 MHz, CDCl.sub.3): 0.93 (3H, t, J=7.2), 0.96
(3H, t, J=7.2), 1.63 (2H, q, J 7.2), 1.7 (2H, q, J=7.2), 2.50-2.54
(2H, m), 2.62-2.66 (2H, m), 4.02 (2H, t, J=6.7), 4.11 (2H, t,
J=6.6), 5.58 (1H, d, J=1.2), 6.19 (1H, s).
[0360] .sup.13C NMR (126 MHz, CDCl.sub.3): 10.35, 10.43, 21.99,
22.00, 27.41, 33.17, 66.01, 66.30, 125.43, 139.25, 166.66,
172.71.
[0361] CI MS: 229 (M+H.sup.+).
[0362] HR CI MS: calcd for C.sub.12H.sub.21O.sub.4 229.1440; found
229.1445.
5-Benzyl 1-(((isopropoxycarbonyl)oxy)methyl)
2-((diphenoxyphosphoryl) methyl)pentanedioate JAM0338
##STR00111##
[0364] The compound JAM0162 (400 mg, 0.819 mmol) was dissolved in
absolute dichloromethane bromotrimethylsilane (0.30 mL, 3.28 mmol,
4 equiv.) was added dropwise and the solution was stirred at
0.degree. C. overnight. The volatiles were removed in vacuo and the
residue was diluted with mixture of acetonitrile and water (5 mL,
4:1) and evaporated. The residue was dissolved in absolute
dichloromethane and catalytic amount of DMF (8 .mu.L) was added. To
the reaction mixture was added oxalyl chloride (0.480 mL, 5.73
mmol, 7 equiv.) and the reaction mixture was stirred at room
temperature for 2 h. The volatiles were removed in vacuo and the
residue was dissolved under argon in absolute dichloromethane (5
mL) and cooled to -20.degree. C. To this mixture was added mixture
of phenol (162 mg, 1.72 mmol, 2.1 equiv.), diisopropylethylamine
(0.5 mL) and pyridine (0.1 mL) in dichloromethane (3 mL). The
reaction mixture was warmed slowly to room temperature and then
stirred for 12 h. The volatiles were removed in vacuo and the
residue was chromatographed on silica gel (hexane-ethyl acetate 2:1
to afford desired product (308 mg, 64%) as an oil.
[0365] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.23 (3H, d, J=2.1), 1.25
(3H, d, J=2.1), 2.09-2.18 (2H, m), 2.18-2.28 (1H, m), 2.39-2.52
(2H, m), 2.56-2.67 (1H, m), 3.07-3.18 (1H, m), 4.85 (1H, hept,
J=6.3), 5.11 (2H, s), 5.70 (1H, d, J=5.7), 5.77 (1H, d, J=5.7),
7.11-7.19 (6H, m), 7.27-7.38 (9H, m).
[0366] .sup.13C NMR (101 MHz, CDCl.sub.3): 21.67 (2C), 27.94 (d,
J.sub.C,P=143.9), 28.19 (d, J.sub.C,P=12.4), 31.24, 39.18 (d,
J.sub.C,P=3.9), 66.63, 73.29, 82.28, 120.58 (2C, d, J.sub.C,P=2.3),
120.62 (2C, d, J.sub.C,P=2.3), 125.42 (d, J.sub.C,P=1.1), 125.44
(d, J.sub.C,P=1.1), 128.39, 128.41, 128.68, 129.33 (2C), 129.94
(2C), 135.82, 150.11 (d, J.sub.C,P=3.4), 150.20 (d, J.sub.C,P=3.6),
153.34, 172.13, 172.29 (d, J.sub.C,P=9.3).
[0367] .sup.31P NMR (101 MHz, CDCl.sub.3): 23.82
[0368] ESI MS: 607 ([M+Na].sup.+).
[0369] HR ESI MS: calcd for C.sub.30H.sub.33O.sub.10NaP 607.17035;
found 607.17038.
4-((diphenoxyphosphoryl)methyl)-5-(((isopropoxycarbonyl)oxy)methoxy)-5-oxo-
pentanoic Acid JAM0338H
##STR00112##
[0371] The same method of preparation as for previous compound
JAM0278R. Phosphonate (300 mg, 0.51 mmol), 10% palladium on carbon
(10 mg), THF (5 mL). Product (247 mg, 98%) as an oil. The product
was further purified using preparative scale HPLC.
[0372] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.26 (3H, d, J=3.9), 1.28
(3H, d, J=3.8), 2.04-2.20 (2H, m), 2.21-2.31 (1H, m), 2.37-2.51
(2H, m), 2.58-2.68 (1H, m), 3.09-3.19 (1H, m), 4.88 (1H, hept,
J=6.3), 5.71 (1H, d, J=5.7), 5.80 (1H, d, J=5.7), 7.12-7.19 (6H,
m), 7.28-7.33 (4H, m).
[0373] .sup.13C NMR (101 MHz, CDCl.sub.3): 21.68 (2C), 27.86 (d,
J.sub.C,P=144.1), 27.95 (d, J.sub.C,P=12.3), 30.86, 39.02 (d,
J.sub.C,P=3.8), 73.38, 82.31, 120.58 (2C, d, J.sub.C,P=1.9), 120.62
(2C, d, J.sub.C,P=1.9), 125.54 (2C), 129.97 (4C), 150.03 (d,
J.sub.C,P=3.4), 150.12 (d, J.sub.C,P=3.6), 153.38, 172.25 (d,
J.sub.C,P=9.5), 176.87.
[0374] .sup.31P NMR (101 MHz, CDCl.sub.3): 24.00
[0375] ESI MS: 517 ([M+Na].sup.+).
[0376] HR ESI MS: calcd for C.sub.23H.sub.28O.sub.10P 495.14146;
found 495.14111.
5-Benzyl 1-(((isopropoxycarbonyl)oxy)methyl)
2-((bis(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)methyl)pentanedio-
ate JAM0341
##STR00113##
[0378] The compound JAM0162 (400 mg, 0.819 mmol) was same method of
preparation as for previous dissolved in absolute dichloromethane
(5 mL) under argon and cooled to 0.degree. C. A
bromotrimethylsilane (0.30 mL, 3.28 mmol, 4 equiv.) was added
dropwise and the solution was stirred at 0.degree. C. overnight.
The volatiles were removed in vacuo and the residue was diluted
with mixture of acetonitrile and water (5 mL, 4: 1) and evaporated.
The residue was dissolved in absolute dichloromethane and catalytic
amount of DMF (8 .mu.L) was added. To the reaction mixture was
added oxalyl chloride (0.480 mL, 5.73 mmol, 7 equiv.) and the
reaction mixture was stirred at room temperature for 2 h. The
volatiles were removed in vacuo and the residue was dissolved under
argon in absolute dichloromethane (5 mL) and cooled to -20.degree.
C. To this mixture was added mixture of L-alanine ethyl ester
hydrochloride (264 mg, 1.72 mmol, 2.1 equiv.),
diisopropylethylamine (1.0 mL) and pyridine (0.1 mL) in
dichloromethane (3 mL). The reaction mixture was warmed slowly to
room temperature and then stirred for 12 h. The volatiles were
removed in vacuo and the residue was chromatographed on silica gel
(chloroform-acetone 5:1 to afford desired product (238 mg, 46%) as
an oil.
[0379] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.15-1.20 (12H, m),
1.27-1.32 (6H, m), 1.67-1.80 (1H, m), 1.88-2.02 (1H, m), 2.08-2.21
(1H, m), 2.31-2.36 (2H, m), 2.83-2.98 (1H, m), 3.07-3.24 (2H, m),
3.85-3.98 (2H, m), 4.00-4.12 (4H, m), 4.80 (1H, hept, J=6.3), 5.02
(2H, s), 5.66 (1H, dd, J=43.8, 5.6), 5.70 (1H, dd, J=67.6, 5.7),
7.21-7.29 (5H, m).
[0380] .sup.31P NMR (101 MHz, CDCl.sub.3): 28.32 and 28.38 (mixture
of diastereoizomers)
4-((bis(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)methyl)-5-(((isopr-
opoxycarbonyl)oxy)methoxy)-5-oxopentanoic Acid JAM0341H
##STR00114##
[0382] The same method of preparation as for previous compound
JAM0278R. Phosphonate (100 mg, 0.16 mmol), 10% palladium on carbon
(5 mg), THF (4 mL). Product (84 mg, 98%) as an oil. The product was
further purified using preparative scale HPLC.
[0383] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.23-1.30 (12H, m),
1.34-1.38 (6H, m), 1.86-2.02 (3H, m), 2.16-2.27 (1H, m), 2.28-2.41
(2H, m), 2.93-3.04 (1H, m), 3.41-3.60 (2H, m), 3.89-4.02 (2H, m),
4.09-4.21 (4H, m), 4.89 (1H, hept, J=6.2), 5.74 (1H, dd, J=41.2,
5.7), 5.76 (1H, dd, J=53.7, 5.7).
[0384] .sup.31P NMR (101 MHz, CDCl.sub.3): 31.00 (both
diastereoizomers)
[0385] ESI MS: 563 ([M+Na].sup.+).
[0386] HR ESI MS: calcd for C.sub.21H.sub.37O.sub.12N.sub.2NaP
563.19763; found 563.19765.
Allyl-3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)propanoate
LTP089
##STR00115##
[0388] Meldrum's acid (5.0 g, 34.69 mmol, 1 eq.), a freshly grinded
K.sub.2CO.sub.3 (4.8 g, 34.69 mmol, 1 eq.) and BnEt.sub.3NCl (7.9
g, 34.69 mmol, 1 eq.) were suspended in dry AcN (50 mL). The
reaction mixture was stirred for 1 hour at room temperature under
inert atmosphere. Allyl acrylate (5.8 g, 6.2 mL, 52.04 mmol, 1.5
eq.) was added and the mixture was heated to 65.degree. C. for 22
hours. AcN was evaporated. The residue was dissolved in EtOAc (200
mL), extracted with 10% KHSO.sub.4 (3.times.150 mL), dried over
MgSO.sub.4 and the solvent was removed by vacuo. The crude product
was suspended in hexane (50 mL) and sonicated. Desired product (7.3
g, 82%) was obtained after filtration as a colorless solid
compound.
[0389] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.76 (3H, s), 1.81 (3H,
s), 2.39 (2H, dt, J=7.2, 5.6), 2.67 (2H, t, J=7.2), 3.91 (1H, t,
J=5.6), 4.57 (2H, dt, J=5.8, 1.4), 5.23 (1H, dq, J=10.4, 1.3), 5.30
(1H, dq, J=17.2, 1.5), 5.90 (1H, ddt, J=17.2, 10.4, 5.8).
[0390] .sup.13C NMR (101 MHz, CDCl.sub.3): 21.30, 26.54, 28.66,
30.33, 44.84, 65.43, 105.23, 118.60, 132.05, 165.23 (2C),
172.61.
t-Butyl-3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)propanoate
LTP096
##STR00116##
[0392] Meldrum's acid (10.0 g, 69.38 mmol, 1 eq.), a freshly
grinded K.sub.2CO.sub.3 (9.6 g, 69.38 mmol, 1 eq.) and
BnEt.sub.3NCl (15.8 g, 69.38 mmol, 1 eq.) were suspended in dry AcN
(100 mL). The reaction 20 mixture was stirred for 1 hour at room
temperature under inert atmosphere. t-Butyl acrylate (13.3 g, 15.1
mL, 104.07 mmol, 1.5 eq.) was added and the mixture was heated to
65.degree. C. for 22 hours. AcN was evaporated. The residue was
dissolved in EtOAc (300 mL), extracted with 10% KHSO.sub.4
(3.times.200 mL), dried over MgSO.sub.4 and the solvent was removed
by vacuo. The crude product was suspended in hexane (100 mL) and
sonicated. Desired product (11.4 g, 60%) was obtained after
filtration as a colorless solid compound.
[0393] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.42 (9H, s), 1.75 (3H,
s), 1.80 (3H, s), 2.28-2.38 (2H, m), 2.52 (2H, td, J=7.3, 0.6),
3.91 (1H, t, J=5.6).
[0394] .sup.13C NMR (101 MHz, CDCl.sub.3): 21.47, 26.57, 28.20
(3C), 28.67, 31.52, 44.93, 80.98, 105.12, 165.35 (2C), 172.25.
Diallyl-2-methylenepentanedioate LTP013
##STR00117##
[0396] A dry Schlenk flask was charged with allyl acrylate (1.00 g,
8.92 mmol, 2 eq.). Tributylphosphine (217 mg, 267 .mu.L, 0.24 eq.)
was added by drop wise. The reaction mixture was stirred for 2 h at
rt under inert (exothermic at the beginning of reaction).
The crude product was purified by column chromatography
(hexane-ethyl acetate 15:1) and the desired product was obtained as
a colourless oil (785 mg) in 79% yield.
[0397] .sup.1H NMR (400 MHz, CDCl.sub.3): 2.54-2.59 (2H, m), 2.67
(2H, t, J=7.4), 4.57 (2H, dt, J=5.8, 1.4), 4.66 (2H, dt, J=5.8,
1.4), 5.15-5.38 (4H, m), 5.62 (1H, d, J=1.2), 5.84-6.01 (2H, m),
6.23 (1H, d, J=1.2).
[0398] .sup.13C NMR (101 MHz, CDCl.sub.3): 27.45, 33.19, 65.31,
65.54, 118.31, 118.43, 126.30, 132.21, 132.27, 138.93, 166.39,
172.45.
5-Allyl-1-t-butyl-2-methylenepentanedioate LTP091
##STR00118##
[0400] A dry Schlenk flask was charged with the compound LTP089
(4.00 g, 15.61 mmol),
[0401] N,N-Dimethylmethyleneiminium iodide (7.22 g, 39.02 mmol, 2.5
eq.) and then it was flushed with argon. Absolute t-BuOH (100 mL)
was added to the flask and the mixture was stirred at 70-75.degree.
C. for 20 h. The organic solvent was evaporated in vacuo. The
residue was dissolved in Et.sub.2O (200 mL) and extracted with sat.
NaHCO.sub.3 (150 mL), 10% KHSO.sub.4 (150 mL), 10%
Na.sub.2S.sub.2O.sub.5 (150 mL), sat. NaCl (150 mL), dried over
MgSO.sub.4. Solvent was evaporated. The crude product was purified
by column chromatography (hexane-ethyl acetate 8:1) to afford the
desired product (3.04 g, 81%) as a colourless oil.
[0402] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.48 (9H, s), 2.49-2.55
(2H, m), 2.56-2.64 (2H, m), 4.56 (2H, dt, J=5.7, 1.4), 5.21 (1H,
dt, J=10.4, 1.2), 5.29 (1H, dt, J=10.4, 1.2), 5.49 (1H, d, J=1.2),
5.83-5.95 (1H, m). 6.08 (1H, d, J=1.2).
[0403] .sup.13C NMR (101 MHz, CDCl.sub.3): 27.53, 28.17, 33.29,
65.22, 80.88, 118.30, 124.87, 132.30, 140.57, 165.99, 172.56.
1-Allyl-5-t-butyl-2-methylenepentanedioate LTP097
##STR00119##
[0405] A dry Schlenk flask was charged with the compound LTP096
(5.00 g, 18.36 mmol),
[0406] N,N-Dimethylmethyleneiminium iodide (8.49 g, 45.91 mmol, 2.5
eq.) and then it was flushed with argon. Absolute allyl alcohol (50
mL) was added to the flask and the mixture was stirred at
70.degree. C. for 20 h. The organic solvent was evaporated in
vacuo. The residue was dissolved in Et.sub.2O (200 mL) and
extracted with sat. NaHCO.sub.3 (150 mL), 10% KHSO.sub.4 (150 mL),
10% Na.sub.2S.sub.2O.sub.5 (150 mL), sat. NaCl (150 mL), dried over
MgSO.sub.4. Solvent was evaporated. The crude product was purified
by column chromatography (hexane-ethyl acetate 10:1) to afford the
desired product (2.37 g, 54%) as a colourless oil.
[0407] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.43 (9H, s), 2.38-2.48
(2H, m), 2.56-2.66 (2H, m), 4.56 (2H, dt, J=5.6, 1.5), 5.24 (1H,
dt, J=10.4, 1.3), 5.33 (1H, dt, J=17.2, 1.6), 5.60 (1H, d, J=1.3),
5.95 (1H, ddt, J=17.2, 10.4, 5.6). 6.21 (1H, d, J=1.3).
[0408] .sup.13C NMR (101 MHz, CDCl.sub.3): 27.57, 28.23, 34.35,
65.47, 80.57, 118.20, 125.87, 132.28, 139.23, 166.51, 172.13.
Diallyl-2-((diethoxyphosphoryl)methyl)pentanedioate LTP016
##STR00120##
[0410] Diethyl phosphite (439 mg, 410 4, 3.18 mmol, 1 eq.) was
dissolved in absolute dichloromethane (8 mL) under argon and cooled
to 0.degree. C. A solution of trimethyl aluminium (2 M in hexanes,
1.59 mL, 3.18 mmol, 1 equiv.) was added dropwise and the solution
was stirred at 0.degree. C. for 30 min. The solution of the
compound LTP013 (712 mg, 3.18 mmol, 1 eq.) in dichloromethane (3
mL) was added during 10 min at 0.degree. C., the mixture was
stirred next 30 min at the same temperature and then the cooling
bath was removed. The reaction mixture was stirred at room
temperature overnight. After 16 h the reaction was quenched with 2
N hydrochloric acid (10 mL). The organic layer was separated and a
water phase was extracted with DCM (2.times.20 mL). The combined
organic layers were washed with water (20 mL), brine (20 mL) and
dried over anhydrous MgSO.sub.4. The crude product was an oil,
which was filtered through pad of silica gel (EtOAc) to afford the
desired product (1.04 g, 90%) as a colourless oil.
[0411] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.24-1.28 (6H, m), 1.81
(1H, ddd, J=18.5, 15.4, 5.3), 1.90-2.02 (2H, m), 2.21 (1H, ddd,
J=18.1, 15.5, 8.5), 2.27-2.39 (2H, m), 2.79 (1H, tdd, J=13.6, 8.3,
5.5), 4.03 (4H, tt, J=8.7, 5.2), 4.53 (4H, dd, J=12.1, 5.7), 5.18
(2H, ddd, J=10.4, 6.8, 1.4), 5.27 (2H, ddd, J=17.2, 12.6, 1.3),
5.79-5.93 (2H, m).
5-Allyl-1-t-butyl-2-((diethoxyphosphoryl)methyl)pentanedioate
LTP093
##STR00121##
[0413] Diethyl phosphite (1.72 g, 1.60 mL, 12.44 mmol, 1 eq.) was
dissolved in absolute dichloromethane (30 mL) under argon and
cooled to 0.degree. C. A solution of trimethyl aluminium (2 M in
hexanes, 6.22 mL, 12.44 mmol, 1 equiv.) was added dropwise and the
solution was stirred at 0.degree. C. for 30 min. The solution of
the compound LTP091 (2.99 g, 12.44 mmol, 1 eq.) in dichloromethane
(10 mL) was added during 15 min at 0.degree. C., the mixture was
stirred next 30 min at the same temperature and then the cooling
bath was removed. The reaction mixture was stirred at room
temperature overnight. After 20 h the reaction was quenched with 2
N hydrochloric acid (40 mL). The organic layer was separated and a
water phase was extracted with DCM (2.times.50 mL). The combined
organic layers were washed with water (20 mL), brine (20 mL) and
dried over anhydrous MgSO.sub.4. The crude product was an oil,
which was filtered through pad of silica gel (EtOAc-hexane 2:1) to
afford the desired product (4.13 g, 88%) as a colourless oil.
[0414] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.28 (6H, td, J=7.1,
1.7), 1.42 (9H, s), 1.69-1.80 (1H, m), 1.84-2.00 (2H, m), 2.12-2.24
(1H, m), 2.26-2.45 (2H, m), 2.59-2.73 (1H, m), 4.06 (4H, dq, J=8.2,
7.1), 4.54 (2H, dt, J=5.7, 1.4), 5.19 (1H, dq, J=10.4, 1.3), 5.27
(1H, dq, J=17.2, 1.5), 5.82-5.93 (1H, m).
1-Allyl-5-t-butyl-2-((diethoxyphosphoryl)methyl)pentanedioate
LTP100
##STR00122##
[0416] Diethyl phosphite (1.36 g, 1.27 mL, 9.86 mmol, 1 eq.) was
dissolved in absolute dichloromethane (26 mL) under argon and
cooled to 0.degree. C. A solution of trimethyl aluminium (2 M in
hexanes, 4.93 mL, 9.86 mmol, 1 equiv.) was added dropwise and the
solution was stirred at 0.degree. C. for 30 min. The solution of
the compound LTP097 (2.37 g, 9.86 mmol, 1 eq.) in dichloromethane
(9 mL) was added during 15 min at 0.degree. C., the mixture was
stirred next 30 min at the same temperature and then the cooling
bath was removed. The reaction mixture was stirred at room
temperature overnight. After 16 h the reaction was quenched with 2
N hydrochloric acid (35 mL). The organic layer was separated and a
water phase was extracted with DCM (2.times.45 mL). The combined
organic layers were washed with water (20 mL), brine (20 mL) and
dried over anhydrous MgSO.sub.4. The crude product was an oil,
which was filtered through pad of silica gel (EtOAc-hexane 2:1) to
afford the desired product (3.20 g, 86%) as a colourless oil.
[0417] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.29 (6H, tdd, J=7.1,
3.3, 0.4), 1.42 (9H, s), 1.78-1.88 (1H, m), 1.89-1.99 (2H, m),
2.16-2.33 (3H, m), 2.75-2.86 (1H, m), 4.01-4.12 (4H, m), 4.59 (2H,
dq, J=5.8, 1.2), 5.23 (1H, dq, J=10.4, 1.3), 5.32 (1H, dq, J=17.2,
1.5), 5.91 (1H, ddt, J=17.2, 10.4, 5.8).
[0418] .sup.13C NMR (101 MHz, CDCl.sub.3): 16.47 (d,
J.sub.C,P=2.0), 16.53 (d, J.sub.C,P=1.8), 37.91 (d,
J.sub.C,P=142.6), 28.18, 28.82 (d, J.sub.C,P=13.0), 32.78, 39.37,
61.83 (d, J.sub.C,P=6.5), 61.92 (d, J.sub.C,P=6.3), 65.66, 80.66,
118.63, 171.87, 173.92 (d, J.sub.C,P=7.9).
5-(Allyloxy)-2-((diethoxyphosphoryl)methyl)-5-oxopentanoic Acid
LTP111
##STR00123##
[0420] Phosphonate LTP093 (1.50 g, 3.96 mmol), was dissolved in
dichloromethane (15 mL) and the mixture was cooled to 0.degree. C.
Trifluoroacetic acid (15 mL) was added slowly during 15 minutes.
The reaction mixture was stirred at room temperature overnight (18
h). Then the solvents were removed in vacuo. The residue was
dissolved in PhCH.sub.3 (2.times.15 mL) and evaporated. The crude
product was purified on silica gel (chloroform-methanol 12: 1) to
furnish the desired product (1.22 g, 95%) as an oil.
[0421] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.31 (6H, td, J=7.1,
2.1), 1.80-2.05 (3H, m), 2.25-2.49 (3H, m), 2.78-2.92 (1H, m),
4.05-4.17 (4H, m), 4.59 (2H, dq, J=5.9, 1.3), 5.25 (1H, dq, J=10.4,
1.2), 5.33 (1H, dq, J=17.2, 1.5), 5.90 (1H, ddt, J=17.2, 10.4,
5.9), 11.40 (1H, bs).
[0422] .sup.13C NMR (101 MHz, CDCl.sub.3): 16.31 (2C, d,
J.sub.C,P=6.2), 27.43 (d, J.sub.C,P=131.9), 28.21, 31.19, 39.02 (d,
J.sub.C,P=3.7), 62.97 (d, J.sub.C,P=5.4), 63.03 (d, J.sub.C,P=5.1),
65.98, 119.07, 131.76, 173.48 (d, J.sub.C,P=8.5), 177.84.
5-(Allyloxy)-4-((diethoxyphosphoryl)methyl)-5-oxopentanoic Acid
LTP104
##STR00124##
[0424] Phosphonate LTP100 (3.19 g, 8.43 mmol), was dissolved in
dichloromethane (30 mL) and the mixture was cooled to 0.degree. C.
Trifluoroacetic acid (30 mL) was added slowly during 20 minutes.
The reaction mixture was stirred at room temperature overnight (16
h). Then the solvents were removed in vacuo. The residue was
dissolved in PhCH.sub.3 (2.times.15 mL) and evaporated. The crude
product was purified on silica gel (chloroform-methanol 12: 1) to
furnish the desired product (2.60 g, 96%) as an oil.
[0425] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.30 (6H, td, J=7.0,
2.1), 1.83-2.07 (3H, m), 2.23-2.36 (1H, m), 2.37-2.49 (2H, m),
2.75-2.87 (1H, m), 4.04-4.19 (4H, m), 4.56 (2H, dt, J 5.8, 1.4),
5.21 (1H, dq, J=10.4, 1.2), 5.29 (1H, dq, J=17.2, 1.5), 5.88 (1H,
ddt, J=17.2, 10.4, 5.8), 12.11 (1H, bs).
[0426] .sup.13C NMR (101 MHz, CDCl.sub.3): 16.21 (d,
J.sub.C,P=2.6), 16.27 (d, J.sub.C,P=2.6), 27.30 (d,
J.sub.C,P=144.2), 28.15 (d, J.sub.C,P=12.6), 31.35, 38.99 (d,
J.sub.C,P=3.7), 62.99 (d, J.sub.C,P=2.5), 63.06 (d, J.sub.C,P=2.4),
65.48, 118.52, 132.03, 172.39, 178.38 (d, J.sub.C,P=8.3).
(5-(Allyloxy)-2-((allyloxy)carbonyl)-5-oxopentyl)phosphonic Acid
LTP018
##STR00125##
[0428] The compound LTP016 (4.02 g, 11.09 mmol) was dissolved in
absolute dichloromethane (62 mL) under argon and cooled to
0.degree. C. Bromotrimethylsilane (6.79 g, 5.86 mL, 44.38 mmol, 4
eq.) was added dropwise during 20 min and the solution was stirred
at 0.degree. C. overnight. The volatiles were removed in vacuo and
the residue was diluted with mixture of acetonitrile and water (30
mL, 4:1) and evaporated. The crude product was obtained in
quantitative yield (3.40 g) and it was used for the next step
without purification.
[0429] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.84-2.02 (3H, m),
2.21-2.36 (1H, m), 2.37-2.43 (2H, m), 2.79-2.92 (1H, m), 4.55-4.64
(4H, m), 5.20-5.27 (2H, m), 5.27-5.32 (1H, m), 5.32-5.37 (1H, m),
5.84-5.97 (2H, m), 9.89 (2H, bs).
5-(Allyloxy)-5-oxo-2-(phosphonomethyl)pentanoic Acid LTP116
##STR00126##
[0431] The compound LTP111 (500 mg, 1.55 mmol) was dissolved in
absolute dichloromethane (10 mL) under argon and cooled to
0.degree. C. Bromotrimethylsilane (1.43 g, 1.23 mL, 9.31 mmol, 6
eq.) was added dropwise during 20 min and the solution was stirred
at 0.degree. C. overnight. The volatiles were removed in vacuo and
the residue was diluted with mixture of acetonitrile and water (20
mL, 4:1) and evaporated. The crude product was obtained in
quantitative yield (413 mg) and it was used for the next step
without purification.
[0432] .sup.1H NMR (400 MHz, d.sup.6-DMSO): 1.56-2.00 (3H, m),
2.24-2.42 (2H, m), 2.52-2.61 (2H, m), 4.53 (2H, dt, J=5.4, 1.6),
5.20 (1H, dq, J=10.3, 1.4), 5.29 (1H, dq, J=17.3, 1.7), 5.90 (1H,
ddt, J=17.3, 10.3, 5.4), 7.53 (3H, bs).
[0433] .sup.13C NMR (101 MHz, d.sup.6-DMSO): 27.62 (d,
J.sub.C,P=9.6), 29.48 (d, J.sub.C,P=137.5), 31.09, 31.26, 64.43,
117.74, 132.78, 172.02, 175.55 (d, J.sub.C,P=10.6).
5-(Allyloxy)-5-oxo-4-(phosphonomethyl)pentanoic Acid LTP115
##STR00127##
[0435] The compound LTP104 (500 mg, 1.55 mmol) was dissolved in
absolute dichloromethane (10 mL) under argon and cooled to
0.degree. C. Bromotrimethylsilane (1.43 g, 1.23 mL, 9.31 mmol, 6
eq.) was added dropwise during 20 min and the solution was stirred
at 0.degree. C. overnight. The volatiles were removed in vacuo and
the residue was diluted with mixture of acetonitrile and water (20
mL, 4:1) and evaporated. The crude product was obtained in
quantitative yield (413 mg) and it was used for the next step
without purification.
[0436] .sup.1H NMR (400 MHz, d.sup.6-DMSO): 1.59-1.81 (2H, m),
1.82-1.99 (2H, m), 2.11-2.28 (2H, m), 2.61-2.72 (1H, m), 4.53 (2H,
dt, J=5.6, 1.5), 5.20 (1H, dq, J=10.5, 1.4), 5.31 (1H, dq, J=17.3,
1.7), 5.91 (1H, ddt, J=17.3, 10.5, 5.6), 7.09 (3H, bs).
[0437] .sup.13C NMR (101 MHz, d.sup.6-DMSO): 28.10 (d,
J.sub.C,P=11.2), 29.72 (d, J.sub.C,P=137.4), 31.12 (2C), 64.67,
117.85, 132.76, 173.72, 173.80 (d, J.sub.C,P=9.1).
Diallyl-2-((bis((4-acetoxybenzyl)oxy)phosphoryl)methyl)pentanedioate
LTP021
##STR00128##
[0439] Compound LTP018 (104 mg, 0.340 mmol, 1 eq.) was dissolved in
dry THF (1.5 mL). Triphenylphosphine (223 mg, 0.849 mmol, 2.5 eq.)
and 4-acetoxybenzyl alcohol (141 mg, 0.849 mmol, 2.5 eq.) was added
in one portion and finally DIAD (172 mg, 167 .mu.L, 0.849 mmol, 2.5
eq.) was added by dropwise during 5 min (exothermic 20 reaction).
The reaction mixture was stirred for 1 h at rt. THF was evaporated
by rotavap and the crude product was purified by column
chromatography (DCM/EtOAc 2:1). The desired product (150 mg, 73%)
was obtained as a viscous colourless oil.
[0440] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.81-2.01 (2H, m), 2.29
(6H, s), 2.30-2.40 (4H, m), 2.75-2.90 (1H, m), 4.37-4.52 (2H, m),
4.55 (2H, d, J=5.4), 4.85-5.06 (4H, m), 5.16-5.42 (4H, m),
5.74-6.01 (2H, m), 7.07 (4H, dd, J=8.6, 2.3), 7.35 (4H, dd, J=7.3,
2.5).
1-(4-Acetoxybenzyl)-5-allyl-2-((bis((4-acetoxybenzyl)oxy)phosphoryl)
Methyl) Pentanedioate LTP119
##STR00129##
[0442] Compound LTP116 (300 mg, 1.13 mmol, 1 eq.) was dissolved in
dry THF (9 mL). Triphenylphosphine (1.18 g, 4.51 mmol, 4 eq.) and
4-acetoxybenzyl alcohol (749 mg, 4.51 mmol, 4 eq.) was added in one
portion and finally DIAD (912 mg, 888 4, 4.51 mmol, 4 eq.) was
added by dropwise during 5 min (exothermic reaction). The reaction
mixture was stirred for 1 h at rt. THF was evaporated by rotavap
and the crude product was purified by column chromatography
(EtOAc/hexane 2:1 to EtOAc). The desired product (275 mg, 34%) was
obtained as a viscous colourless oil.
[0443] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.82-2.02 (3H, m),
2.25-2.39 (3H, m), 2.30 (3H, s), 2.31 (6H, s), 2.79-2.91 (1H, m),
4.55 (2H, dt, J=5.7, 1.4), 4.88-5.04 (6H, m), 5.22 (1H, dq, J=10.4,
1.3), 5.29 (1H, dq, J=17.2, 1.5), 5.89 (1H, ddt, J=17.2, 10.4,
5.7), 7.03-7.11 (6H, m), 7.29-7.37 (6H, m).
5-(4-Acetoxybenzyl)-1-allyl-2-((bis((4-acetoxybenzyl)oxy)phosphoryl)
Methyl) Pentanedioate LTP122
##STR00130##
[0445] Compound LTP122 (400 mg, 1.50 mmol, 1 eq.) was dissolved in
dry THF (12 mL). Triphenylphosphine (1.58 g, 6.01 mmol, 4 eq.) and
4-acetoxybenzyl alcohol (999 mg, 6.01 mmol, 4 eq.) was added in one
portion and finally DIAD (1.22 g, 1.18 mL, 6.01 mmol, 4 eq.) was
added by dropwise during 5 min (exothermic reaction). The reaction
mixture was stirred for 1 h at rt. THF was evaporated by rotavap
and the crude product was purified by column chromatography
(EtOAc). The desired product (1.0 g, 94%) was obtained as a viscous
colourless oil.
[0446] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.76-2.00 (3H, m),
2.22-2.38 (3H, m), 2.27 (3H, s), 2.28 (6H, s), 2.73-2.86 (1H, m),
4.43 (2H, dt, J=5.8, 1.6), 4.85-5.02 (6H, m), 5.17 (1H, dq, J=10.3,
1.2), 5.24 (1H, dq, J=17.2, 1.5), 5.89 (1H, ddt, J=17.2, 10.3,
5.8), 7.02-7.09 (6H, m), 7.28-7.38 (6H, m).
[0447] .sup.13C NMR (101 MHz, CDCl.sub.3): 21.05, 21.11 (2C),
21.68, 21.90, 21.96, 28.15 (d, J.sub.C,P=142.2), 28.39 (d,
J.sub.C,P=13.5), 31.32, 39.11 (d, J.sub.C,P=3.7), 64.53, 65.64,
65.71, 66.74 (d, J.sub.C,P=2.5), 66.80 (d, J.sub.C,P=2.1), 69.99,
118.63, 121.59, 121.73, 121.81 (d, J.sub.C,P=2.5), 128.00, 129.24,
129.49, 131.79, 133.42, 133.63 (d, J.sub.C,P=1.8), 133.69 (d,
J.sub.C,P=1.4), 138.73, 149.95, 150.53, 150.71 (d, J.sub.C,P=1.7),
169.30, 169.40, 169.58, 172.14, 173.40 (d, J.sub.C,P=7.8).
2-((Bis((4-acetoxybenzyl)oxy)phosphoryl)methyl)pentanedioic Acid
LTP023
##STR00131##
[0449] The starting material LTP021 (144 mg, 0.239 mmol, 1 eq.) was
dissolved in dry THF (2 mL). Pd(PPh.sub.3).sub.4 (13.8 mg, 0.012
mmol, 5 mol %) was added in one portion and finally phenyl silane
(103 mg, 117 .mu.L, 0.956 mmol, 4 eq.) was added by dropwise during
2 min. The reaction mixture was stirred at rt under inert
atmosphere for 1 h. THF was evaporated and crude product was
filtered through pad of silica gel (EtOAc/MeOH 2:1) and finally
purified by preparative HPLC. The desired product (50 mg, 40%) was
obtained as an amorphous solid (after lyophilisation).
[0450] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.70-2.04 (2H, m), 2.28
(6H, s), 2.31-2.49 (4H, m), 2.70-2.81 (1H, m), 4.85-5.04 (4H, m),
7.05 (4H, dd, J=8.4, 1.6), 7.32 (4H, dd, J=8.5, 1.6), 7.81 (2H,
s).
5-(4-Acetoxybenzyl)-4-((bis((4-acetoxybenzyl)oxy)phosphoryl)methyl)-5-oxop-
entanoic Acid LTP120
##STR00132##
[0452] The starting material LTP119 (139 mg, 0.196 mmol, 1 eq.) was
dissolved in dry THF (2 mL). Pd(PPh.sub.3).sub.4 (11.3 mg, 0.010
mmol, 5 mol %) was added in one portion and finally phenyl silane
(42 mg, 48 .mu.l, 0.391 mmol, 2 eq.) was added by dropwise during 2
min. The reaction mixture was stirred at rt under inert atmosphere
for 1 h. THF was evaporated and crude product was filtered through
pad of silica gel (CHCl.sub.3/MeOH 20:1 to 10:1) and finally
purified by preparative HPLC. The desired product (114 mg, 87%) was
obtained as an oil.
[0453] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.82-1.97 (2H, m), 2.29
(3H, s), 2.30 (6H, s), 2.16-2.37 (4H, m), 2.72-2.85 (1H, m),
4.86-5.03 (6H, m), 6.03 (1H, bs), 7.00-7.08 (6H, m), 7.28-7.35 (6H,
m).
[0454] .sup.13C NMR (101 MHz, CDCl.sub.3): 21.25 (3C), 27.97 (d,
J.sub.C,P=142.8), 28.13 (d, J.sub.C,P=13.1), 30.80, 38.98 (d,
J.sub.C,P=3.6), 66.35, 67.32 (d, J.sub.C,P=3.7), 67.38 (d,
J.sub.C,P=3.6), 121.88 (2C), 122.05 (2C), 122.06 (2C), 129.55 (2C),
129.60 (2C), 129.78 (2C), 133.22, 133.51, 133.57, 150.73, 150.91,
150.93, 169.59, 169.67, 169.74, 173.51 (d, J.sub.C,P=8.5),
175.36.
5-(4-Acetoxybenzyl)-2-((bis((4-acetoxybenzyl)oxy)phosphoryl)methyl)-5-oxop-
entanoic Acid LTP123
##STR00133##
[0456] The starting material LTP122 (500 mg, 0.703 mmol, 1 eq.) was
dissolved in dry THF (4 mL). Pd(PPh.sub.3).sub.4 (41 mg, 0.035
mmol, 5 mol %) was added in one portion and finally phenyl silane
(152 mg, 173 .mu.L, 1.41 mmol, 2 eq.) was added by dropwise during
2 min. The reaction mixture was stirred at rt under inert
atmosphere for 1 h. THF was evaporated and crude product was
filtered through pad of silica gel (CHCl.sub.3/MeOH 15:1) and
finally purified by preparative HPLC. The desired product (246 mg,
52%) was obtained as an oil.
[0457] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.83-2.04 (2H, m), 2.27
(6H, s), 2.28 (3H, s), 2.30-2.47 (4H, m), 2.72-2.83 (1H, m),
4.88-5.03 (4H, m), 5.05 (2H, s), 7.05 (6H, dd, J=8.5, 2.0), 7.32
(6H, dd, J=8.1, 1.4), 9.24 (1H, bs).
[0458] .sup.13C NMR (101 MHz, CDCl.sub.3): 21.14 (2C), 21.15, 27.89
(d, J.sub.C,P=143.0), 28.22 (d, J.sub.C,P=13.8), 31.35, 38.79 (d,
J.sub.C,P=3.5), 65.79, 67.47, 67.54, 121.77 (2C), 121.94 (4C),
129.43 (2C), 129.44 (2C), 129.52 (2C), 133.32, 133.38, 133.49,
150.56, 150.85 (2C), 169.52, 169.53, 169.57, 172.40, 176.57 (d,
J.sub.C,P=7.5).
1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
5-(2-(trimethylsilyl)ethyl)
2-((diethoxyphosphoryl)methyl)pentanedioate TT-160614
##STR00134##
[0460] To a solution of
2-((diethoxyphosphoryl)methyl)-5-oxo-5-(2-(trimethylsilyl)ethoxy)pentanoi-
c acid (1.49 g, 3.90 mmol),
4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (663 mg, 5.1 mmol) and
HOBt (690 mg, 5.1 mmol) in anhydrous DMF (10 ml) a solution of
EDC.HCl (980 mg, 5.1 mmol) and DMAP (623 mg, 5.1 mmol) in DMF (10
ml) was added. The mixture was stirred at RT overnight. The solvent
was evaporated and the residue was extracted with EtOAc --H.sub.2O
mixture (1:1, 200 ml). Aqueous portion was extracted with EtOAc (50
ml) again. Organic extracts were dried with MgSO.sub.4 and
evaporated. The residue was chromatographed on a silica gel column
in EtOAc.fwdarw.1% MeOH/EtOAc. Yield 1.75 g of oil (91%).
[0461] .sup.1H NMR (400 MHz, CDCl.sub.3): 0.04 (s, 9H,
Si--CH.sub.3); 0.98 (m, 2H, Si--CH.sub.2); 1.29-1.33 (m, 6H,
CH.sub.3(Et)), 1.88 (ddd, 1H, Jib-13=18.7, J.sub.gem=15.4,
J.sub.1b-2=5.0, H-1b), 1.95-2.03 (m, 2H, H-3), 2.19 (t, 3H,
J.sub.CH3-CH2=0.5, 4'--CH.sub.3), 2.21 (ddd, J.sub.1a-P=17.8,
J.sub.gem=15.4, J.sub.1a-2=8.9, H-1a), 2.27-2.35 (m, 2H, H-4), 2.84
(m, 1H, H-2), 4.05-4.12 (m, 4H, CH.sub.2 (Et)), 4.16 (m, 2H,
OCH.sub.2 (TMSE)), 4.84 (dq, 1H, J.sub.gem=13.9, J.sub.CH2-CH3=0.5,
3'-CH.sub.2b), 4.89 (dq, 1H, J.sub.gem=13.9, J.sub.CH2-CH3=0.5,
3'-CH.sub.2a).
[0462] .sup.13C NMR (101 MHz, CDCl.sub.3): -1.55 (Si--CH.sub.3),
9.37 (4'-CH.sub.3), 16.37 (d, J.sub.CH3-P=6.0, CH.sub.3(Et)), 17.26
(Si--CH.sub.2), 27.69 (d, J.sub.1-P=128.7, C-1), 28.31 (C-3), 31.41
(C-4), 39.15 (d, J.sub.2-P=3.7, C-2), 54.17 (3'-CH.sub.2),
61.81-61.92 (m, CH.sub.2 (Et)), 62.90 (OCH.sub.2(TMSE)), 133.24
(C-3'), 140.20 (C-4'), 152.01 (C-1'), 171.40 (C-5), 173.48 (d,
J.sub.C-2-1-P=7.7, COO).
[0463] ESI MS: 517 ([M+Na].sup.+).
[0464] HR ESI MS: calcd for C.sub.20H.sub.35O.sub.10NaPSi
517.16293; found 517.16306.
1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
5-(2-(trimethylsilyl)ethyl)
2-((bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)pentane-
dioate TT-230614
##STR00135##
[0466] To a solution of 1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
5-(2-(trimethylsilyl)ethyl)
2-((diethoxyphosphoryl)methyl)pentanedioate (1.70 g, 3.438 mmol) in
anhydrous acetonitrile (40 ml) Me.sub.3SiBr (3.6 ml, 27.5 mmol) was
added at 0.degree. C. The mixture was kept at 0.degree. C.
overnight, then evaporated and codistilled with MeCN, dissolved in
dioxane (30 ml) and treated with water (240 .quadrature.l, 13.3
mmol). The solution was stirred 30 min at room temperature then
toluen (30 ml) was added and the solvents were evaporated. To a
solution of resulting phosphonic acid, triphenylphosphine (5.4 g,
20.6 mmol) and 4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (2.68 g,
20.6 mmol) in THF (80 ml) DIAD (4.0 ml, 20.6 mmol) was added
dropwise. The mixture was stirred 6 h at room temperature. The
solvent was removed in vacuo and the residue was chromatographed on
a silica gel column in EtOAc.fwdarw.3% MeOH/EtOAc. Yield 1.01 g
(44%).
[0467] .sup.1H NMR (400 MHz, CDCl.sub.3): 0.04 (s, 9H,
Si--CH.sub.3), 0.98 (m, 2H, Si--CH.sub.2), 1.94-2.02 (m, 3H, H-3,
H-1b), 2.20 (3.times.s, 9H, 4'-CH.sub.3), 2.25-2.34 (m, 3H, H-1a,
H-4), 2.83 (m, 1H, H-2), 4.16 (m, 2H, OCH.sub.2(TMSE)), 4.77-4.93
(m, 6H, 3'-CH.sub.2).
[0468] .sup.13C NMR (101 MHz, CDCl.sub.3): -1.56 (Si--CH.sub.3),
9.30, 9.33 (4'-CH.sub.3), 17.24 (Si--CH.sub.2), 28.15 (d,
J.sub.1-P=141.5, C-1), 28.26 (d, J.sub.3-P=14.5, C-3), 31.16 (C-4),
38.76 (d, J.sub.2-P=3.8, C-2), 54.37 (COO--CH.sub.2-3'), 55.23,
55.35 (2.times.d, J.sub.C-O-P=5.8, 6.0, P--O--CH.sub.2), 63.02
(OCH.sub.2 (TMSE)), 132.96 (C-3'), 133.29, 133.30 (2.times.d,
J.sub.3'-P=6.0, C-3'), 140.30, 140.31, 140.44 (C-4'), 151.71,
151.74, 152.07 (C-1'), 172.24 (C-5), 172.93 (d, J.sub.C-2-1-P=6.6,
COO).
[0469] ESI MS: 685 ([M+Na].sup.+).
[0470] HR ESI MS: calcd for C.sub.26H.sub.35O.sub.16NaPSi
685.13242; found 685.13262.
4-((hydroxy((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)-5-(-
(5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-5-oxopentanoic Acid
TT-200714
##STR00136##
[0472] A solution of 1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
5-(2-(trimethylsilyl)ethyl)
2-((bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)
pentanedioate (690 mg, 1.04 mmol) in anhydrous dichloromethane (10
ml) was treated with TFA (1 ml) at 0.degree. C. The mixture was
stirred 48 h at 0.degree. C. then evaporated and the residue was
chromatographed twice on a silica gel column in EtOAc.fwdarw.50%
MeOH/EtOAc. Yield 110 mg (23%).
[0473] .sup.1H NMR (400 MHz, CD3COCD.sub.3): 1.80-2.12 (m, 4H, H-3,
H-1), 2.21 (bs, 6H, 2.times.CH.sub.3), 2.32, 2.40 (2.times.bs, 2H,
H-4), 2.90 (bs, 1H, H-2), 4.74-5.09 (m, 4H, O--CH.sub.2). .sup.13C
NMR (101 MHz, CD3COCD.sub.3): 9.34, 9.43 (2.times.CH.sub.3), 29.0
(C-1, C-3), 31.7 (C-4), 40.1 (C-2), 54.92, 55.22
(2.times.OCH.sub.2), 134.69, 136.26 (C-3'), 139.88, 141.21 (C-4'),
152.97, 153.22 (C-1'), 172.15 (C-5), 175.50 (COO).
[0474] ESI MS: 449 ([M-H].sup.-).
[0475] HR ESI MS: calcd for C.sub.16H.sub.18O.sub.13P 449.04905;
found 449.04895.
Bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)2-((diethoxyphosphoryl)methyl)
Pentanedioate TT-301014
##STR00137##
[0477] Dibenzyl 2-((diethoxyphosphoryl)methyl)pentanedioate (2.31
g, 5.0 mmol) was hydrogenated in anhydrous THF (100 ml) in the
presence of catalytic amount of 10% Pd/C at room temperature and 1
atm overnight. The catalyst was filtered off and
4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (1.49 g, 11.0 mmol),
HOBt (1.49 g, 11.0 mmol) were added. A solution of EDC.HCl (2.11 g,
11.0 mmol) and DMAP (1.34 g, 11.0 mmol) in DMF (60 ml) was added
and the mixture was stirred at room temperature overnight. Solvents
were evaporated and the residue was partitioned between
EtOAc--H.sub.2O (1:1, 200 ml). The aqueous solution was extracted
with EtOAc (100 ml) again and combined organic extracts were dried
(MgSO4) and evaporated. The residue was chromatographed on a silica
gel column in EtOAc.fwdarw.2% MeOH/EtOAc. Yield 2.13 g (84%) of
oil.
[0478] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.30-1.33 (m, 6H,
CH.sub.3(Et)), 1.87 (ddd, 1H, J.sub.1b-P=18.6, J.sub.gem=15.4,
J.sub.1b-2=5.5, H-1b), 1.96-2.08 (m, 2H, H-3), 2.18, 2.19
(2.times.s, 2.times.3H, 4'-CH.sub.3), 2.21 (ddd, J.sub.1a-P=18.1,
J.sub.gem=15.4, J.sub.1a-2=8.5, H-1a), 2.32-2.44 (m, 2H, H-4), 2.84
(m, 1H, H-2), 4.05-4.13 (m, 4H, CH.sub.2 (Et)), 4.82-4.93 (m, 4H,
3'-CH.sub.2).
[0479] .sup.13C NMR (101 MHz, CDCl.sub.3): 9.35, 9.37
(4'-CH.sub.3), 16.36, 16.37 (2.times.d, J.sub.CH3-P=5.9,
CH.sub.3(Et)), 27.72 (d, J.sub.1-P=143.2, C-1), 27.86 (d,
J.sub.3-P=12.6, C-3), 30.88 (C-4), 38.96 (d, J.sub.2-P=3.5, C-2),
53.84, 54.22 (3'-CH.sub.2), 61.91-62.00 (m, CH.sub.2 (Et)), 133.15,
133.30 (C-3'), 140.17, 140.30 (C-4'), 152.01, 152.05 (C-1'), 171.72
(C-5), 173.29 (d, J.sub.C-2-1-P=8.6, COO).
[0480] ESI MS: 529 ([M+Na].sup.+).
[0481] HR ESI MS: calcd for C.sub.20H.sub.27O.sub.13NaP 529.10815;
found 529.10819.
(5-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-2-(((5-methyl-2-oxo-1,3-dioxo-
l-4-yl)methoxy)carbonyl)-5-oxopentyl)phosphonic Acid TT-120814
##STR00138##
[0483] A solution of bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
2-((diethoxyphosphoryl)methyl) pentanedioate (253 mg, 0.5 mmol) in
anhydrous acetonitrile (5 ml) was treated with Me3SiBr (530 .mu.l,
4.0 mmol) at 0.degree. C. The solution was kept at 0.degree. C.
overnight. The volatiles were evaporated and the residue was
codistilled with MeCN and treated with water (0.25 ml). The sample
was purified on a C-18 reverse phase column in gradient
H.sub.2O.fwdarw.55% MeOH/H.sub.2O. Yield 170 mg (75%).
[0484] ESI MS: 449 ([M -H].sup.-).
[0485] HR ESI MS: calcd for C.sub.16H.sub.18O.sub.13P 449.04905;
found 449.04877.
Bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
2-((bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)pentane-
dioate TT-190215
##STR00139##
[0487] A solution of bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
2-((diethoxyphosphoryl)methyl) pentanedioate (250 mg, 0.494 mmol)
in anhydrous acetonitrile (5 ml) was treated with Me.sub.3SiBr (530
.mu.l, 4.0 mmol) at 0.degree. C. The solution was kept at 0.degree.
C. overnight. The volatiles were evaporated and the residue was
codistilled with MeCN, dissolved in dioxane (5 ml) and treated with
water (36 .mu.l, 2.0 mmol). The solution was stirred 30 min at room
temperature then toluen (5 ml) was added and the solvents were
evaporated. To a solution of resulting phosphonic acid,
triphenylphosphine (777 mg, 2.96 mmol) and
4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (386 mg, 2.96 mmol) in
THF (10 ml, DIAD (583 .mu.l, 2.96 mmol) was added dropwise. The
mixture was stirred 6 h at room temperature. The solvent was
removed in vacuo and the residue was chromatographed on a silica
gel column in EtOAc.fwdarw.4% MeOH/EtOAc. Yield 160 mg (48%).
[0488] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.93-2.01 (m, 3H, H-3,
H-1b), 2.19, 2.20 (3.times.s, 3H, 6H, 3H, 4.times.CH.sub.3),
2.24-2.45 (m, 3H, H-4, H-1a), 2.82 (m, 1H, H-2), 4.77-4.96 (m, 8H,
O--CH.sub.2).
[0489] .sup.13C NMR (101 MHz, CDCl.sub.3): 9.24, 9.27, 9.29
(4.times.CH.sub.3), 27.90 (d, J3-P=14.0, C-3), 28.07 (d,
J1-P=141.8, C-1), 30.67 (C-4), 38.51 (d, J2-P=3.7, C-2), 53.88,
54.40 (2.times.OCH2), 55.23, 55.37 (2.times.d, JCH2-O--P=6.0 and
5.8, CH.sub.2--O--P), 132.89-133.30 (m, C-3'), 140.23, 140.34,
140.51 (C-4'), 151.72, 151.75, 152.05, 152.07 (C-1'), 171.49 (C-5),
172.74 (d, JC-P=7.4, COO).
[0490] ESI MS: 697 ([M+Na].sup.+).
[0491] HR ESI MS: calcd for C.sub.26H.sub.27O.sub.19NaP 697.07764;
found 697.07752.
bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
2-((hydroxy((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)pen-
tanedioate TT-110814
##STR00140##
[0493] A solution of bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)
2-((bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)
phosphoryl)methyl)pentanedioate (256 mg, 0.38 mmol) and LiN3 (37
mg, 1.07 mmol) in DMF (2 ml) was stirred at room temperature
overnight. The solvent was evaporated and the residue was purified
on a silica gel column in 10-50% MeOH/EtOAc. Yield 136 mg
(64%).
[0494] .sup.1H NMR (400 MHz, CDCl.sub.3): 1.74-2.07 (m, 4H, H-3,
H-1), 2.16, 2.18, 2.18 (3.times.s, 9H, 3.times.CH.sub.3), 2.26-2.41
(m, 2H, H-4), 2.78 (m, 1H, H-2), 4.69-4.96 (m, 6H,
O--CH.sub.2).
[0495] .sup.13C NMR (101 MHz, CDCl.sub.3): 9.02, 9.20, 9.22
(3.times.CH.sub.3), 27.80-29.13 (C-1, C-3), 30.78 (C-4), 39.62
(C-2), 53.92, 54.28, 54.44 (3.times.OCH.sub.2), 133.23, 133.33
(C-3'), 134.94 (d, J.sub.3'-C-O-P=7.0, C-3'), 139.23, 140.39,
140.48 (C-4'), 152.22, 152.29, 152.46 (C-1''), 172.57 (C-5), 174.78
(m, COO).
[0496] ESI MS: 561 ([M-H].sup.-)
[0497] HR ESI MS: calcd for C.sub.21H.sub.22O.sub.16P 561.06509;
found 561.06496.
Example 3
Selective Deprotection of Phosphonate Diesters
##STR00141##
[0499] Bromotrimethylsilane (9.3 mL; 70 mmol) was added at
0.degree. C. to a solution of appropriate phosphonate diester (17.5
mmol) in acetonitrile (100 mL) and kept at 0.degree. C. for 24 h.
The solution was evaporated, the residue coevaporated with
acetonitrile, followed by water and toluene. The crude product was
purified by chromatography on silica gel in system chloroform-ethyl
acetate-methanol (2:2:1).
[0500] The following compounds were prepared:
[5-(Methyloxy)-2-(methoxycarbonyl)-5-oxopentyl]phosphonic Acid.
TT-140113
[0501] Yield: 3.2 g (73%) of a colorless syrup. .sup.1H NMR
(CDCl.sub.3, ppm) 6: 1.84 (m, 1H, H-1a); 1.97 (m, 2H, H-3); 2.15
(m, 1H, H-1b); 2.36 (m, 2H, H-4); 2.79 (m, 1H, H-2); 3.66 (s, 3H,
R.sub.1=Me); 3.69 (s, 3H, R.sub.2=Me). .sup.13C NMR (CDCl.sub.3,
ppm) 6: 29.53 (d, J.sub.C,P=12.8, C-3); 30.33 (d, J.sub.C,P=140.4,
C-1); 32.10 (C-4); 40.89 (d, J.sub.C,P=3.4, C-2); 52.43
(R.sub.1=Me); 52.47 (R.sub.2=Me); 174.83 (COOR.sub.1); 176.44 (d,
J.sub.C,P=8.1, COOR.sub.2). ESI MS, m/z:
[5-(Benzyloxy)-2-(methoxycarbonyl)-5-oxopentyl]phosphonic Acid.
TT-100313
[0502] Yield: 4.5 g (78%) of a colorless syrup. The crude product
was used for the preparation of compound TT-110313 without
purification and identification.
[5-(Benzyloxy)-2-(ethoxycarbonyl)-5-oxopentyl]phosphonic Acid.
MK-800
[0503] Yield: 4.0 g (66%) of a white waxy solid. ESI MS, m/z: 687.3
[2M-H].sup.- (43), 343.1 [M-H].sup.-(50). HRMS (ESI): For
C.sub.15H.sub.20O.sub.7P [M-H].sup.- calculated: 343.09521; found:
34309513.
[5-Ethoxy-2-(ethoxycarbonyl)-5-oxopentyl]phosphonic Acid.
MK-797
[0504] Yield: 3.0 g (60%) of a colorless syrup. .sup.31P{.sup.1H}
NMR (CDCl.sub.3, ppm) .delta.: 26.70. NMR (CDCl.sub.3, ppm) 6: 1.29
(m, 6H, 2.times.CH.sub.3, Et), 2.06 (m, 3H), 2.27 (m, 3H), 2.83 (m,
1H, H-2), 4.14 (m, 4H, 2.times.CH.sub.2, Et). ESI MS, m/z: 563.3
[2M-H].sup.- (100), 281.2 [M-H].sup.- (62). HRMS (ESI): For
C.sub.10H.sub.18O.sub.7P [M-H].sup.- calculated: 281.07956; found:
281.07958. Anal. Calcd. for C.sub.10H.sub.19O.sub.7P: C, 42.56; H,
6.79; P, 10.97. Found: C, 42.07; H, 6.87; P, 10.59.
[5-Oxo-5-propoxy-2-(propoxycarbonyl)pentyl]phosphonic Acid.
MK-801
[0505] Yield: 3.6 g (67%) of a white waxy solid. .sup.31P{.sup.1H}
NMR (CD.sub.3OD, ppm) .delta.: 21.38. .sup.1H NMR (CD.sub.3OD, ppm)
.delta.: 0.92 (m, 6H, 2.times.CH.sub.3, Pr), 1.39 (m, 2H), 1.67 (m,
3H), 2.09 (m, 3H), 2.31 (m, 2H), 2.88 (m, 1H), 4.16 (m, 4H). ESI
MS, m/z: 619.3 [2M-H].sup.-(100), 309.1 [M-H].sup.- (47). HRMS
(ESI): For C.sub.12H.sub.22O.sub.7P [M-H].sup.- calculated:
309.11086; found: 309.11101.
[5-(Benzyloxy)-2-(benzyloxycarbonyl)-5-oxopentyl]phosphonic Acid.
MK-824
[0506] Yield: 6.0 g (85%) of a colorless syrup. ESI MS, m/z: 811.3
[2M-H].sup.- (12), 405.1 [M-H].sup.- (26).
Example 4
Esterification of Phosphonic Acids --POM Esters, POC Esters, Alkyl
Esters
##STR00142##
[0508] DBU (2 mmol) was added to a solution of appropriate
phosphonic acid (1 mmol) in dry dioxane (10 mL) and then heated
with POC-Cl (20 mmol, 80.degree. C., 4 h) or POM-Cl (4 mmol,
reflux, 6 h) or decyl bromide (2.3 mmol, reflux, 3 h) or
3,6,9,12,15,18-hexaoxaicosyl p-toluensulfonate (2.1 mmol, reflux, 6
h), respectively. The reaction course was monitored by TLC in
system toluene-acetone (4:1); detection was performed by spraying
of the TLC plate with a solution of phosphomolybdenic acid and
heating. Reaction mixture was evaporated and the residue
chromatographed on a silica gel column (200 mL) in system
toluene-acetone (4:1) for POM and POC esters, toluene-acetone (8:1)
for decyl ester, or 12% MeOH/CHCl.sub.3 for
3,6,9,12,15,18-hexaoxaicosyl ester.
[0509] The following compounds were prepared:
5-Benzyl 1-methyl
2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-pentanedioate-
. MK-792
[0510] Yield: 194 mg (34%) of a colourless syrup. .sup.31P{.sup.1H}
NMR (CDCl.sub.3, ppm) .delta.: 29.85. .sup.1H NMR (CDCl.sub.3, ppm)
.delta.: 1.31-1.33 (m, 12H, CH.sub.3 iPr), 1.96-2.06 (m, 3H, H-3,
H-1b), 2.34-2.44 (m, 3H, H-4, H-1a), 2.84 (m, 1H, H-2), 3.69 (s,
3H, COOCH.sub.3), 4.93 (2.times. septet, 2H, J.sub.CH,CH3=6.3,
2.times.CH iPr), 5.11 (s, 2H, CH.sub.2Bn), 5.60-5.68 (m, 4H,
2.times.OCH.sub.2O), 7.30-7.38 (m, 5H, H-2',3',4'). .sup.13C NMR
(CDCl.sub.3, ppm) .delta.: 21.59 (CH.sub.3 iPr), 28.16 (d,
J.sub.3,P=13.6, C-3), 28.54 (d, J.sub.1,P=143.2, C-1), 31.34 (C-4),
38.65 (d, J.sub.2,P=3.6, C-2), 52.15 (OCH.sub.3), 66.41 (CH.sub.2
Bn), 73.27 (CH iPr), 83.94 and 83.95 (2.times.d, J.sub.C-O-P=6.2,
OCH.sub.2O), 128.25 (C-2'), 128.26 (C-4'), 128.54 (C-3'), 135.76
(C-1'), 153.11 and 153.13 (OC(O)O), 172.12 (C-5), 173.82 (d,
J.sub.C-C-C-P=8.7, 2--COO). ESI MS, m/z: 585.5 [M+Na].sup.+ (100),
563.5 [MH].sup.+ (31). HRMS (ESI): For C.sub.24H.sub.36O.sub.13P
[MH].sup.+ calculated: 563.18880; found: 563.18868.
5-Benzyl 1-methyl 2-((bis(decyloxy)phosphoryl)methyl)pentanedioate.
MK-796
[0511] Yield: 351 mg (57%) of a colourless syrup. .sup.31P
{.sup.1H} NMR (CDCl.sub.3, ppm) .delta.: 28.39. .sup.1H NMR
(CDCl.sub.3, ppm) .delta.: 0.90 (7, 6H, J.sub.CH3,CH2=6.9,
2.times.CH.sub.3, decyl), 1.30 (m, 28H, 14.times.CH.sub.2, decyl),
1.65 (m, 4H, CH.sub.2), 1.86 (ddd, 1H), 2.03 (m, 2H), 2.26 (ddd,
1H), 2.41 (m, 2H), 2.83 (tdd, 1H, H-2), 3.71 (s, 3H, COOCH.sub.3),
4.00 (qd, 4H, 2.times.OCH.sub.2, decyl), 5.13 (s, 2H, CH.sub.2,
Bn), 7.36 (m, 5H, H-arom.). ESI MS, m/z: 1243.8 [2M+Na].sup.+ (38),
633.4 [M+Na].sup.+ (100). HRMS (ESI): For
C.sub.34H.sub.59O.sub.7PNa [M+Na].sup.+ calculated: 633.38906;
found: 633.38806.
Diethyl
2-({bis[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate.
MK-798
[0512] Yield: 429 mg (84%) of a yellowish syrup. .sup.31P {.sup.1H}
NMR (CDCl.sub.3, ppm) .delta.: 29.46. .sup.1H NMR (CDCl.sub.3, ppm)
.delta.: 1.25 (m, 24H, 8.times.CH.sub.3, POM, Et), 1.99 (m, 3H),
2.34 (m, 3H), 2.80 (m, 1H, H-2), 4.17 (m, 4H, 2.times.CH.sub.2,
Et), 5.67 (m, 4H, 2.times.OCH.sub.2O). ESI MS, m/z: 533.1
[M+Na].sup.+ (100). HRMS (ESI): For C.sub.22H.sub.39O.sub.11PNa
[M+Na].sup.+ calculated: 533.21222; found: 533.21221. Anal. Calcd.
for C.sub.22H.sub.39O.sub.11P: C, 51.76; H, 7.70; P, 6.07. Found:
C, 51.98; H, 7.55; P, 6.01.
5-Benzyl 1-ethyl
2-({bis[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate.
MK-803
[0513] Yield: 183 mg (32%) of a colourless syrup. .sup.31P NMR
(CDCl.sub.3, ppm) .delta.: 29.40. .sup.1H NMR (CDCl.sub.3, ppm)
.delta.: 1.25 (m, 21H, 7.times.CH.sub.3, POM, Et), 2.01 (m, 3H),
2.39 (m, 3H), 2.82 (m, 1H, H-2), 4.17 (q, 2H, J.sub.CH2,CH3=7.1,
CH.sub.2, Et), 5.13 (s, 2H, CH.sub.2, Bn), 5.66 (d, 2H, J=13.1,
OCH.sub.2O), 5.67 (d, 2H, J=13.1, OCH.sub.2O), 7.36 (m, 5H,
H-arom.). ESI MS, m/z: 595.3 [M+Na].sup.+ (100). HRMS (ESI): For
C.sub.27H.sub.41O.sub.11PNa [M+Na].sup.+ calculated: 595.22787;
found: 595.22783.
5-Benzyl 1-ethyl
2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-pentanedioate-
. MK-805
[0514] Yield: 217 mg (38%) of a colourless syrup. .sup.31P{.sup.1H}
NMR (CDCl.sub.3, ppm) .delta.: 29.46. .sup.1H NMR (CDCl.sub.3, ppm)
.delta.: 1.27 (t, 3H, J.sub.CH3,CH2=7.1, CH.sub.3, Et), 1.33 (d,
6H, J.sub.CH3,CH=1.6, 2.times.CH.sub.3, iPr), 1.34 (d, 6H,
J.sub.CH3,CH=1.6, 2.times.CH.sub.3, iPr), 2.04 (m, 3H), 2.41 (m,
3H), 2.83 (m, 1H, H-2), 4.17 (q, 2H, J.sub.CH2,CH3=7.1, CH.sub.2,
Et), 5.13 (s, 2H, CH.sub.2, Bn), 5.66 (m, 4H, 2.times.OCH.sub.2O,
POC), 7.36 (m, 5H, H-arom.). ESI MS, m/z: 1175.7 [2M+Na].sup.+ (6),
599.3 [M+Na].sup.+ (100). HRMS (ESI): For
C.sub.25H.sub.38O.sub.13PNa [M+Na].sup.+ calculated: 577.20445;
found: 577.20468.
Dibenzyl
2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)pentan-
edioate. MK-825
[0515] Yield: 247 mg (39%) of a colourless syrup. .sup.31P{.sup.1H}
NMR (CDCl.sub.3, ppm) .delta.: 29.31. .sup.1H NMR (CDCl.sub.3, ppm)
.delta.: 1.34 (4.times.d, 12H, 4.times.CH.sub.3, POC), 2.04 (m,
3H), 2.37 (m, 3H), 2.90 (m, 1H, H-2), 4.93 (m, 2H, 2.times.CH,
POC), 5.11 (s, 2H, CH.sub.2, Bn), 5.14 (d, 2H, CH.sub.2, Bn), 5.63
(m, 4H, 2.times.OCH.sub.2O), 7.35 (m, 10H, H-arom.). ESI MS, m/z:
661.2 [M+Na].sup.+ (100).
Dimethyl
2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-penta-
nedioate. TT-010213
[0516] Yield: 177 mg (37%) of a colourless syrup. .sup.1H NMR
(CDCl.sub.3, ppm) .delta.: 1.31 (4.times.d, 12H, 4.times.Me, POM);
1.98 (m, 2H, H-3); 2.00 (ddd, 1H, J.sub.1a,P=19.1, J.sub.gem=15.6,
J.sub.1a,2=5.4, H-1a); 2.32 (m, 2H, H-4); 2.37 (ddd, 1H,
J.sub.1b,P=19.0, J.sub.gem=15.6, J.sub.1a,28.6, H-1b); 2.82 (m, 1H,
H-2); 3.65 (s, 3H, Me, R.sub.1); 3.70 (s, 3H, Me, R.sub.2); 4.92
(2.times.sept, 2H, 2.times.CH, POC); 5.58-5.68 (m, 4H,
2.times.CH.sub.2, POC). .sup.13C NMR (CDCl.sub.3, ppm) .delta.:
21.58 (4.times.CH.sub.3, POC); 28.13 (d, J.sub.C,P=13.5, C-3);
28.45 (d, J.sub.C,P=143.3, C-1); 31.09 (d, J.sub.C,P=1.1, C-4);
38.62 (d, J.sub.C,P=3.6, C-2); 51.69 (Me, R.sub.1); 52.17 (Me,
R.sub.2); 73.28 (CH, POC); 83.91, 83.92 (2.times.d, J.sub.C,P=6.2,
2.times.CH.sub.2, POC); 153.10, 153.12 (2.times.CO, POC); 172.74
(COOR.sub.1); 173.83 (d, J.sub.C,P=8.9, COOR.sub.2). ESI MS,
m/z:
Dimethyl
2-((bis{[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate.
TT-201212A
[0517] Yield: 195 mg (40%) of a colourless syrup. .sup.1H NMR
(CDCl.sub.3, ppm) .delta.: 1.22 (2.times.s, 2.times.9H, 6.times.Me,
POM); 1.91-2.03 (m, 3H, H-1a+H-3); 2.26-2.38 (m, 3H, H-1b+H-4);
2.80 (dtt, 1H, J.sub.2,P=13.5, J.sub.2,1b=J.sub.2,3b=8.4,
J.sub.2,1a=J.sub.2,3a=5.5, H-2); 3.65 (s, 3H, Me, R.sub.1); 3.70
(s, 3H, Me, R.sub.2); 5.61-5.67 (m, 4H, 2.times.CH.sub.2, POM).
.sup.13C NMR (CDCl.sub.3, ppm) .delta.: 26.79 (6.times.CH.sub.3,
POM); 28.14 (d, J.sub.C,P=13.3, C-3); 28.69 (d, J.sub.C,P=142.8,
C-1); 31.07 (d, J.sub.C,P=1.1, C-4); 38.70 (C, POM); 38.71 (d,
J.sub.C,P=3.5, C-2); 51.71 (Me, R.sub.1); 52.17 (Me, R.sub.2);
81.36, 81.39 (2.times.d, J.sub.C,P=6.2, 2.times.CH.sub.2, POM);
172.72 (COOR.sub.1); 173.87 (d, J.sub.C,P=9.1, COOR.sub.2). 176.80,
176.81 (2.times.CO, POM). ESI MS, m/z: 505.2 [M+Na].sup.+ (100).
HRMS (ESI): For C.sub.20H.sub.35O.sub.11PNa [M+Na].sup.+
calculated: 505.18092; found: 505.18099.
Dimethyl
2-((bis((3,6,9,12,15,18-hexaoxaicosyl)oxy)phosphoryl)methyl)penta-
nedioate TT-250113
[0518] Yield: 342 mg (41%) of a colourless syrup. .sup.1H NMR
(CDCl.sub.3, ppm) .delta.: 1.20 (t, 6H, J.sub.14',13'=7.0, H-14');
1.94 (ddd, 1H, J.sub.1a,P=18.8, J.sub.gem=15.5, J.sub.1a,2=5.6,
H-1a); 1.98 (m, 2H, H-3); 2.28 (ddd, 1H, J.sub.1b,P=18.5,
J.sub.gem=15.5, J.sub.1b,2=8.4, H-1b); 2.33 (m, 2H, H-4); 2.82 (m,
1H, H-2); 3.51 (q, 4H, J.sub.13',14'=7.0, H-13'); 3.56-3.68 (m,
44H, H-2' to H-12'); 3.65 (s, 3H, Me, R.sub.1); 3.69 (s, 3H, Me,
R.sub.2); 4.15 (m, 4H, H-1'). .sup.13C NMR (CDCl.sub.3, ppm)
.delta.: 15.11 (C-14'); 27.65 (d, J.sub.C,P=143.3, C-1); 28.17 (d,
J.sub.C,P=12.6, C-3); 31.16 (d, J.sub.C,P=1.0, C-4); 38.99 (d,
J.sub.C,P=3.6, C-2); 51.66 (Me, R.sub.1); 52.04 (Me, R.sub.2);
64.71, 64.76 (2.times.d, J.sub.C,P=5.9, 2.times.C-1'); 66.60
(C-13'); 70.60-69.77 (m, C-2' to C-12'); 170.90 (COOR.sub.1);
174.32 (d, J.sub.C,P=9.2, COOR.sub.2). ESI MS, m/z: 861.5
[M+Na].sup.+ (100). HRMS (ESI): For C.sub.36H.sub.72O.sub.19P
[M+H].sup.+ calculated: 839.43999; found: 839.44023.
Example 5
Esterification of Phosphonic Acids --Alkyl Salicylyl Esters
##STR00143##
[0520] Catalytic amount of DMF (10 .mu.L), followed by oxalyl
chloride (0.6 mL; 7 mmol) were added to a solution of phosphonic
acid (1 mmol) in dry dichloromethane (10 mL). The mixture was
stirred for 2 h and evaporated. The residue (intermediary
phosphochloridate) was dissolved in dichloromethane (5 mL), cooled
to -10.degree. C. and dry pyridine (0.16 mL) was added dropwise.
The resulting mixture was immediately added to a cooled
(-30.degree. C.) mixture of butyl salicylate (0.4 g; 2.1 mmol) and
triethyl amine (0.85 mL) in dichloromethane (8 mL). The reaction
mixture was warmed slowly to room temperature, then stirred for 12
h and evaporated. The residue was chromatographed on a column of
silica gel (80 mL) in system toluene-acetone (10:1).
The following compounds were prepared:
5-Benzyl 1-methyl
2-({bis[2-(butoxycarbonyl)phenoxy]phosphoryl}methyl)pentane-dioate.
MK-794
[0521] Yield: 0.41 g (60%) of a yellowish syrup. .sup.31P{.sup.1H}
NMR (CDCl.sub.3, ppm) .delta.: 23.45. .sup.1H NMR (400 MHz,
CDCl.sub.3, ppm) .delta.: 0.965 (t, 3H, J.sub.CH3,CH2=7.4,
CH.sub.3), 0.968 (t, 3H, J.sub.CH3,CH2=7.4, CH.sub.3), 1.46 (m, 4H,
CH.sub.2), 1.73 (m, 4H, CH.sub.2), 2.18 (m, 2H), 2.51 (m, 3H), 2.80
(m, 1H), 3.18 (m, 1H), 3.66 (s, 3H, COOCH.sub.3), 4.29 (2.times.t,
4H, J.sub.CH2,CH2=6.7, OCH.sub.2 (Bu)), 5.11 (s, 2H, CH.sub.2(Bn)),
7.23 (m, 4H, H-arom.), 7.34 (m, 5H, H-arom.), 7.40 (m, 2H,
H-arom.), 7.88 (m, 2H, H-arom.). ESI MS, m/z: 1387.3 [2M+Na].sup.+
(12), 705.1 [M+Na].sup.+ (100), 683.1 [MH].sup.+ (48). HRMS (ESI):
For C.sub.36H.sub.43O.sub.11PNa [M+Na].sup.+ calculated: 705.24352;
found: 705.24280.
Dimethyl
2-({bis[2-(butoxycarbonyl)phenoxy]phosphoryl}methyl)pentanedioate-
. TT-100113
[0522] Yield: 0.48 g (79%) of a yellowish syrup. .sup.1H NMR
(CDCl.sub.3, ppm) .delta.: 0.95 (2.times.t, 6H, J.sub.4'',3''=7.4,
H-4''); 1.43 (m, 4H, H-3''); 1.68-1.74 (m, 4H, H-2''); 2.13 (m, 2H,
H-3); 2.41 (m, 2H, H-4); 2.50 (ddd, 1H, J.sub.1a,P=19.8,
J.sub.gem=15.5, 6.1, H-1a); 2.79 (ddd, 1H, J.sub.1b,P=19.4,
J.sub.gem=15.5, J.sub.1b,2=7.7, H-1b); 3.18 (m, 1H, H-2); 3.64 (s,
3H, Me, R.sub.1); 3.65 (s, 3H, Me, R.sub.2); 4.28 (2.times.t, 4H,
J.sub.1'',2''=6.8, H-1''); 7.19 (m, 2H, H-5'); 7.21 (m, 2H, H-3');
7.39 (m, 2H, H-4'); 7.86 (m, 2H, H-6'). .sup.13C NMR (CDCl.sub.3,
ppm) .delta.: 13.70 (C-4''); 19.18 (C-3''); 28.17 (d,
J.sub.C,P=12.4, C-3); 28.37 (d, J.sub.C,P=144.7, C-1); 30.63
(C-2''); 31.24 (d, J.sub.C,P=0.8, C-4); 38.76 (d, J.sub.C,P=3.4,
C-2); 51.59 (Me, R.sub.1); 52.04 (Me, R.sub.2); 65.03, 65.05
(C-1''); 122.49, 122.54 (2.times.d, J.sub.C,P=0.9, C-3'); 123.36,
123.40 (C-1'); 124.90, 124.91 (2.times.d, J.sub.C,P=1.4, C-5');
133.52, 133.55 (2.times.d, J.sub.C,P=0.6, C-6'); 133.34, 133.35
(C-4'); 149.12, 149.13 (2.times.d, J.sub.C,P=9.4, C-2'); 164.64,
164.67 (2.times.d, J.sub.C,P=1.1; COOBu); 172.99 (COOR.sub.1);
174.20 (d, J.sub.C,P=11.0, COOR.sub.2). ESI MS, m/z: 629.3
[M+Na].sup.+ (100). HRMS (ESI): For C.sub.30H.sub.39O.sub.11PNa
[M+Na].sup.+calculated: 629.21222; found: 629.21169.
Example 6
Synthesis of Bis Amidates
##STR00144##
[0524] Catalytic amount of DMF (10 .mu.L), followed by oxalyl
chloride (0.6 mL; 7 mmol) were added to a solution of phosphonic
acid (1 mmol) in dry dichloromethane (10 mL). The mixture was
stirred for 2 h and evaporated. The residue (intermediary
phosphochloridate) was dissolved in dichloromethane (5 mL), cooled
to -10.degree. C. and dry pyridine (0.16 mL) was added dropwise.
The resulting mixture was immediately added to a cooled
(-30.degree. C.) mixture of 1-phenylalanine ethyl ester
hydrochloride (0.48 g; 2.1 mmol) and triethyl amine (0.85 mL) in
dichloromethane (8 mL). The reaction mixture was warmed slowly to
room temperature, then stirred overnight and finally washed with an
aqueous solution of citric acid (20 ml). Organic portion was dried
and concentrated. The residue was chromatographed on a column of
silica gel (80 mL) in system MeOH--EtOAc--CHCl.sub.3 (4:50:50).
[0525] The following compound was prepared:
Dimethyl
2-((bis(((S)-1-ethoxy-1-oxo-3-phenylpropan-2-yl)amino)phosphoryl)-
methyl)pentanedioate TT-280113
[0526] Yield: 0.39 g (48%) of a yellowish syrup (mixture of
diastereomers). .sup.1H NMR (CDCl.sub.3, ppm) .delta.: 1.20, 1.21,
1.23 (4.times.t, 6H, J.sub.2'',1''=7.1, H-2''); 1.19, 1.35
(2.times.m, 1H, H-1a); 1.73-1.98 (m, 3H, H-1b+H-3); 2.13-2.32 (m,
2H, H-4); 2.59, 2.73 (m, 1H, H-2); 2.77, 2.85, 2.95, 3.07
(4.times.m, 4H, H-3'); 3.58, 3.61 (2.times.s, 3H, Me, R.sub.2);
3.65, 3.66 (2.times.s, 3H, Me, R.sub.1); 3.95, 4.04 (2.times.m, 2H,
H-1''); 4.07-4.28 (m, 4H, H-2'+H-1''); 7.10, 7.18 (2.times.m, 4H,
H-5'); 7.20-7.32 (m, 6H, H-6'+H-7'). .sup.13C NMR (CDCl.sub.3, ppm)
.delta.: 14.04, 14.05, 14.08 (C-2''); 28.69, 28.81 (2.times.d,
J.sub.C,P=14.0 and 15.0, C-3); 30.91 (2.times.d, J.sub.C,P=115.9,
C-1); 31.01 (C-4); 39.11, 39.25 (2.times.d, J.sub.C,P=3.3 and 3.6,
C-2); 40.30, 40.46, 40.81, 40.93 (4.times.d, J.sub.C,P=4.4, 4.5,
4.9 and 5.4, C-3'); 51.66, 51.67 (Me, R.sub.1); 52.07, 52.13 (Me,
R.sub.2); 53.56, 53.94, 53.96, 54.37 (C-2'); 61.21, 61.22, 61.25,
61.33 (C-1''); 126.82, 126.87, 126.90, 126.91 (C-7'); 128.36,
128.41, 128.42, 128.44 (C-6'); 129.47, 129.59, 129.65, 129.69
(C-5'); 136.30, 136.37, 136.64, 136.67 (C-4'); 172.95, 172.98,
173.00, 173.02 (COOR.sub.1); 173.16, 173.22 (2.times.d,
J.sub.C,P=3.1 and 2.4, C-1'); 175.03, 175.45 (2.times.d,
J.sub.C,P=4.8 and 4.1, COOR.sub.2). ESI MS, m/z: 626.9 [M+Na].sup.+
(100). HRMS (ESI): For C.sub.30H.sub.41O.sub.9N.sub.2PNa
[M+Na].sup.+ calculated: 627.24419; found: 627.24396.
Example 7
Synthesis of Monoesters
##STR00145##
[0528] 10% Pd/C (90 mg) was added to a solution of benzyl ester (1
mmol) in THF (30 mL) and the mixture was hydrogenated at room
temperature and atmospheric pressure for 15 h. The catalyst was
removed by filtration through a pad of Celite. The crude filtrate
was finally purified by additional filtration through a Whatman
nylon membrane filter. The filtrate was evaporated to give a
desired monoester as a colorless syrup in yield 90-100%. The
reaction course was monitored by TLC in system ethyl
acetate-acetone-ethanol-water (18:3:2:2), detection was performed
by spraying with bromocresol green solution and heating (white spot
of the product).
[0529] The following products were prepared:
4-({Bis[(pivaloyloxy)methoxy]phosphoryl}methyl)-5-methoxy-5-oxopentanoic
acid. TT 150313
[0530] .sup.1H NMR (400 MHz, CDCl.sub.3, ppm) .delta.: 1.25 (s,
18H, 6.times.CH.sub.3), 2.02 (m, 3H), 2.38 (m, 3H), 2.85 (m, 1H,
H-2), 3.73 (s, 3H, COOCH.sub.3), 5.67 (d, 4H, J=13.1,
2.times.OCH.sub.2O).
4-((Bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-5-methoxy-5-ox-
opentanoic Acid. Mk-793
[0531] .sup.31P{.sup.1H} NMR (CDCl.sub.3, ppm) .delta.: 30.01.
.sup.1H NMR (400 MHz, CDCl.sub.3, ppm) .delta.: 1.32-1.34 (m, 12H,
CH.sub.3), 1.98-2.08 (m, 3H, H-3, H-1b), 2.33-2.44 (m, 3H, H-4,
H-1a), 2.86 (m, 1H, H-2), 3.72 (s, 3H, COOCH.sub.3), 4.94
(2.times.sept, 2H, OCH(CH.sub.3).sub.2), 5.61-5.69 (m, 4H,
OCH.sub.2O). .sup.13C NMR (CDCl.sub.3, ppm) .delta.: 21.59
(CH.sub.3), 27.85 (d, J.sub.3,P=12,9, C-3), 28.40 (d,
J.sub.1,P=143.3, C-1), 30.94 (C-4), 38.51 (d, J.sub.2,P=3.6, C-2),
52.21 (OCH.sub.3), 73.52 (CH iPr), 84.02 and 84.03 (2.times.d,
J.sub.C,P=6.3, OCH.sub.2O), 153.12 and 153.15 (O(CO)O), 173.83 (d,
J.sub.C,P=9.6, COOMe), 176.38 (COOH). ESI MS, m/z: 495.4
[M+Na].sup.+ (100), 473.4 [MH].sup.+ (58). HRMS (ESI): For
C.sub.17H.sub.29O.sub.13PNa [M+Na].sup.+ calculated: 495.12380;
found: 495.12378.
4-({Bis[2-(butoxycarbonyl)phenoxy]phosphoryl}methyl)-5-methoxy-5-oxopentan-
oic Acid MK-795
[0532] .sup.31P{.sup.1H} NMR (CDCl.sub.3, ppm) .delta.: 24.12.
.sup.1H NMR (400 MHz, CDCl.sub.3, ppm) .delta.: 0.95 (m, 6H,
CH.sub.3), 1.44 (m, 4H, CH.sub.2CH.sub.3), 1.72 (m, 4H,
CH.sub.2CH.sub.2CH.sub.3), 2.05-2.19 (m, 2H, H-3), 2.39-2.57 (m,
3H, H-1a, H-4), 2.82 (ddd, 1H, J.sub.1b,P=19.6, J.sub.gem=15.6,
J.sub.1b,2=7.6, H-1b), 3.20 (m, 1H, H-2), 3.66 (s, 3H, OCH.sub.3),
4.27-4.31 (m, 4H, COOCH.sub.2), 7.19-7.25 (m, 4H, H-arom.), 7.40
(m, 2H, H-arom.), 7.87 (m, 2H, H-arom.). .sup.13C NMR (CDCl.sub.3,
ppm) .delta.: 13.61 (CH.sub.3), 19.11 (CH.sub.2CH.sub.3), 27.85 (d,
J.sub.3,P=11.9, C-3), 28.28 (d, J.sub.1,P=144.8, C-1), 30.58
(CH.sub.2CH.sub.2CH.sub.3), 31.12 (C-4), 38.62 (d, J.sub.2,P=3.1,
C-2), 51.96 (OCH.sub.3), 65.01 and 65.02 (2.times.OCH.sub.2),
122.43-122.50 (m, C-3'), 123.31-123.37 (m, C-1'), 124.91 (C-5'),
131.48 and 131.50 (C-6'), 133.32 (C-4'), 149.01-149.10 (m, C-2'),
164.59 and 164.62 (COOBu), 174.08 (d, J.sub.C,P=11.5, COOMe),
177.32 (COOH). ESI MS, m/z: 615.2 [M+Na].sup.+ (100). HRMS (ESI):
For C.sub.29H.sub.37O.sub.11PNa [M+Na].sup.+ calculated: 615.19657;
found: 615.19577.
4-{[Bis(decyloxy)phosphoryl]methyl}-5-methoxy-5-oxopentanoic Acid.
Mk-799
[0533] .sup.31P{.sup.1H} NMR (CDCl.sub.3, ppm) .delta.: 29.45.
.sup.1H NMR (400 MHz, CDCl.sub.3, ppm) .delta.: 0.88 (t, 6H,
J.sub.CH3,CH2=7.1, CH.sub.3 (H-10')), 1.23-1.36 (m, 28H, CH.sub.2
(H-3'-H-9')), 1.61-1.67 (m, 4H, CH.sub.2 (H-2')), 1.90 (ddd, 1H,
J.sub.1a,P=18.5, J.sub.gem=15.5, J.sub.1a,2=5.5, H-1a), 1.95-2.05
(m, 2H, H-3), 2.26 (ddd, 1H, J.sub.1b,P=18.3, J.sub.gem=15.5,
J.sub.1b,2=8.2, H-1b), 2.31-2.43 (m, 2H, H-4), 2.84 (m, 1H, H-2),
3.70 (s, 3H, COOCH.sub.3), 3.98-4.02 (m, 4H, CH.sub.2 (H-1')).
.sup.13C NMR (CDCl.sub.3, ppm) .delta.: 14.09 (C-10'), 22.65
(C-9'), 25.47 (C-3'), 27.45 (d, J.sub.1,P=142.9, C-1), 28.14 (d,
J.sub.3,P=12.1, C-3), 29.16 (C-4'), 29.28 and 29.51 (C-5',6',7'),
30.46 (d, J.sub.2',P=6.1, C-2'), 31.12 (C-4), 31.87 (C-8'), 38.92
(d, J.sub.2,P=3.5, C-2), 52.01 (OCH.sub.3), 66.05-66.11 (m, C-1'),
174.41 (d, J.sub.C,P=9.1, COOMe), 176.11 (COOH). -ESI MS, m/z:
1039.8 [2M-H].sup.-(10), 519.4 [M-H].sup.- (100). HRMS (-ESI): For
C.sub.27H.sub.52O.sub.7P [M-H].sup.- calculated: 519.34561; found:
519.34549.
4-({Bis[(pivaloyloxy)methoxy]phosphoryl}methyl)-5-ethoxy-5-oxopentanoic
Acid. MK-804
[0534] .sup.31P{.sup.1H} NMR (CDCl.sub.3, ppm) .delta.: 29.56.
.sup.1H NMR (400 MHz, CDCl.sub.3, ppm) .delta.: 1.25 (m, 21H,
CH.sub.3), 2.01 (m, 3H), 2.37 (m, 3H), 2.83 (ddd, 1H), 4.18 (m, 2H,
CH.sub.2CH.sub.3), 5.68 (d, 4H, J=12.9, OCH.sub.2O). ESI MS, m/z:
505.1 [M+Na].sup.+ (100). HRMS (ESI): For
C.sub.20H.sub.35O.sub.11PNa [M+Na].sup.+ calculated: 505.18092;
found: 505.18109.
4-((Bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-5-ethoxy-5-oxo-
pentanoic Acid. Mk-806
[0535] .sup.31P{.sup.1H} NMR (CDCl.sub.3, ppm) .delta.: 29.72.
.sup.1H NMR (400 MHz, CDCl.sub.3, ppm) .delta.: 1.30 (m, 15H,
CH.sub.3), 2.03 (m, 3H), 2.40 (m, 3H), 2.85 (m, 1H), 4.18 (q, 2H,
J.sub.CH2,CH3=7.1, CH.sub.2CH.sub.3), 4.94 (dq, 2H, J=12.4 and 6.2,
OCH(CH.sub.3).sub.2), 5.61-5.75 (m, 4H, OCH.sub.2O). ESI MS, m/z:
509.0 [M+Na].sup.+ (100). HRMS (ESI): For
C.sub.18H.sub.31O.sub.13PNa [M+Na].sup.+ calculated: 509.13945;
found: 509.13962.
Example 8
Synthesis of Dicarboxylic Acid
##STR00146##
[0537] Catalytic amount of DMF (10 .mu.L), followed by oxalyl
chloride (0.6 mL; 7 mmol) were added to a solution of compound
MK-824 (1 mmol) in dry dichloromethane (10 mL). The mixture was
stirred for 2 h and evaporated. The residue (intermediary
phosphochloridate) was dissolved in dichloromethane (5 mL), cooled
to -10.degree. C. and dry pyridine (0.16 mL) was added dropwise.
The resulting mixture was immediately added to a cooled
(-30.degree. C.) mixture of hexadecyloxypropyl alcohol (0.63 g; 2.1
mmol) and triethyl amine (0.85 mL) in dichloromethane (8 mL). The
reaction mixture was warmed slowly to room temperature, then
stirred for 12 h and evaporated. The residue was chromatographed on
a column of silica gel (80 mL) in system toluene-acetone (10:1).
The fractions containing phosphonic ester intermediate were
evaporated (580 mg, 60%), the residue was hydrogenated in THF (30
mL) in the presence of 10% Pd/C (cat.) at atmospheric pressure for
24 h. The catalyst was removed by filtration through a pad of
celite. The crude filtrate was finally purified by additional
filtration through a Whatman nylon membrane filter. The filtrate
was evaporated to give a desired monoester which was crystallized
from hexane in freezer (-20.degree. C.).
[0538] The following compound was prepared:
2-((Bis(3-(hexadecyloxy)propoxy)phosphoryl)methyl)pentanedioic
Acid. TT-041212
[0539] Yield 670 mg (85%) of crystals. .sup.1H NMR (CDCl.sub.3,
ppm) .delta.: 0.87 (t, 6H, J.sub.19',18'=7.0, H-19'); 1.19-1.35 (m,
52H, H-6' to H-18'); 1.54 (m, 4H, H-5'); 1.90 (m, 1H, H-1a); 1.91
(m, 4H, H-2'); 2.04 (m, 2H, H-3); 2.30 (ddd, 1H, J.sub.1b,P=18.2,
J.sub.gem=15.6, J.sub.1b,2=8.5, H-1b); 2.47 (m, 2H, H-4); 2.83 (m,
1H, H-2); 3.39 (t, 4H, J.sub.4',5'=6.8, H-4'); 3.48 (m, 4H, H-3');
4.12 (m, 4H, H-1'). .sup.13C NMR (CDCl.sub.3, ppm) .delta.: 14.11
(C-19'); 22.68 (C-18'); 26.13 (C-6'); 27.24 (d, J.sub.C,P=142.3,
C-1); 28.04 (d, J.sub.C,P=12.6, C-3); 29.70-29.36 (m, C-5'+C-7' to
C-16'); 30.64, 30.66 (2.times.d, J.sub.C,P=6.4, C-2'); 31.43 (C-4);
31.91 (C-17'); 39.11 (bd, J.sub.C,P=3.3, C-2); 63.42 (d,
J.sub.C,P=6.5, C-1'); 66.46 (C-3'); 71.23 (C-4'); 177.19 (C-5);
178.06 (d, J.sub.C,P=8.4, C-6). Elem. An. for
C.sub.44H.sub.87O.sub.9P calc.: C 66.80, H 11.08, P 3.92; found: C
66.93, H 11.18, P 3.79.
##STR00147##
Example 9
Simple Alkyl Esters are not Effective 2PMPA Prodrugs
[0540] In general, the prodrugs were screened by an in vitro
metabolic stability screen, and if positive, followed by an in vivo
single time point pharmacokinetic study, and in select cases, an in
vivo full time course pharmacokinetic study (FIG. 3). In this
Example, the four acidic functional groups in 2-PMPA were
systematically capped. The carboxylic acids were first masked with
simple alkyl esters (Compounds 1, 2, and 3). In vitro chemical and
metabolic stability of the prodrugs then were conducted, following
60 min incubation in plasma stability screens for prodrugs. Simple
carboxylic esters like 1, 2 and 3, unexpectedly, turned out to be
too stable, likely due to a very hydrophilic nature of the
phophonate containing part of their molecules (FIG. 4 shows the
stability screen for compound 1).
Example 10
Capping Both Phosphonates and Alpha Carboxylate Critical for
Enhancing Permeability
[0541] Masking of the phosphonate while keeping the carboxylates
free as in the bis-isopropyloxycarbonyl methyl derivative (bis-POC,
4) and bis-pivaloyloxy methyl derivative (bis POM, 5) below alone
is not feasible because of the chemical instability of those
derivatives. The likely cause is direct participation of the
.alpha.-carboxylate in the hydrolysis of the POC or POM group.
Combination of both approaches as illustrated by example below
renders compounds 6 and 7 with good compound penetrability. These
compounds are, however, only converted to the corresponding
carboxylate ester 1 which is stable in plasma and did not show the
ability to release 2-PMPA. Very similar results were obtained with
corresponding diethyl esters. In vitro metabolic stability of the
2-PMPA prodrugs 6 and 7 also were conducted. These prodrugs were
found to be stable to chemical hydrolysis and unstable in mouse
plasma and mouse liver microsomes (FIG. 5 shows the stability
screen for compound 6).
[0542] Single time point pharmacokinetic studies were then
performed on the 2-PMPA prodrugs to evaluate for enhancement in
prodrug permeability and PMPA release. FIG. 6 shows the
concentrations of 6, 7 and 8 tested and their comparison to 2-PMPA
following oral administration at 30 mg/kg equivalent dose of
2-PMPA. Compounds 6 and 7 showed 25-50 fold enhancement in
permeability when compared to 2-PMPA alone. More importantly, 8
with a free .gamma.-carboxylate also showed similar enhancement in
permeability. However no release of 2-PMPA was observed from any of
these prodrugs and thus further optimization was needed. Since it
is the .alpha.-carboxylate responsible for the instability of
bis-POC and bis-POM compounds 4 and 5, derivatization of the
.gamma.-carboxylate was unnecessary as prodrugs with free
.gamma.-carboxylate 8 and 9 also exhibited good oral availability.
But even in this case, the bioconversion only proceeded mostly to
monoester 10. This was also the case of corresponding ethyl esters
(not shown).
Example 11
Pivaloyloxymethyl (POM) and Propyloxycarbonyloxymethyl (POC) on
Alpha Carboxylate Found to be Critical for Enhancement of
Permeability and Release of Free 2PMPA
[0543] In vitro metabolic stability screens of compounds JHU 2106
and 2108-2112 in mouse plasma and liver subcellular fractions are
shown in FIGS. 7-9 and Tables 2-7. Compound JHU 2106, comprising
methyl esters, was found to be too stable and therefore hindered
the release of 2-PMPA (FIG. 7A, Table 2). Compound JHU 2108 was
also found to be too stable in some of the samples (FIG. 7B, Table
3). Compound JHU 2109 was found to fall apart easily even in HBSS
buffer and therefore would not even be able to get metabolized
(FIG. 8A, Table 4). Compounds JHU 2110, 2111, and 2112 were found
to be stable in HBSS buffer and were able to release 2-PMPA
efficiently (FIGS. 8B, 9A-9B; Tables 5-7). An in vivo single time
point pharmacokinetic study (method 1) of compounds JHU 2106-2112
in mice suggested that the POM (JHU 2109) and POC (JHU 2110)
prodrugs were the most permeable (FIG. 10). A more than 50-fold
increase of the POM and POC ester prodrugs/metabolites was seen
following oral dosing (method 1; FIG. 7). However, the POM and POC
ester prodrugs did not release 2-PMPA (method 2; FIG. 8).
Increasing the ester chain length on the carboxylates did not
increase 2-PMPA release (FIGS. 9A-9B, Tables 8-9). No or minimal
2-PMPA release was observed with the ethyl and propyl esters.
Compounds 2236 and 2237, ethyl and alkyl ester derivatives, were
found to be too stable and not much release of 2-PMPA was observed
(Tables 8-9).
[0544] However, the stability of the simple carboxylic ester could
be overcome by introducing another isopropyloxycarbonyloxy methyl
or pivaloyloxy methyl moiety on the .alpha.-carboxylate. The
Tris-POC (JAM0186) and Tris-POM (JAM0168) prodrugs demonstrated
sufficient chemical stability, especially the POC moiety, and
instability in plasma and liver subcellular fractions depicting the
potential of releasing 2-PMPA (FIGS. 10A-10B; scheme for synthesis
of Tris-POC shown in FIG. 1). Without wishing to be bound to any
one particular theory, it is believed that the double esters on the
Tris-POC prodrug allow better release of the prodrug to 2-PMPA. The
POM esters on the carboxylate increased 2-PMPA approximately
18-fold following oral dosing. In vivo pharmacokinetic studies at
30 mg/kg equivalent 2-PMPA in mice showed about a 20-fold increase
in the 2-PMPA availability (FIG. 10B). This is the first time high
micromolar concentrations of 2-PMPA have been achieved in plasma
following oral administration.
[0545] In addition, compound 2609, with an extra methyl group, also
demonstrated sufficient chemical stability, and instability in
plasma and liver subcellular fractions (Table 10).
[0546] In terms of permeability of the esters on the
.alpha.-carboxylate, the ethyl ester (compound JHU 2236) showed the
most permeability, followed by the methyl ester (compound JHU
2106), and then the propyl ester (compound JHU 2263). Even so,
compounds with simple alkyl esters on the .alpha.-carboxylate,
though they showed some enhancement in permeability, were too
stable to cause the release of 2-PMPA in vivo. In addition, both
the mono and diesters showed comparable permeabilities and not much
difference was observed between the mono ethyl and mono methyl
esters. Except for the POM and POC esters on phosphonates
(compounds JAM0168 and JAM0186), most of the other functionalities
did not show a high permeability in vivo (e.g., compounds JHU 2235
and 2238). The POM and POC esters on phosphonate showed the highest
permeability and were chosen as appropriate promoeities for
phosphonic acid and further structural modification were based on
these for enhancement of 2-PMPA permeability and release.
TABLE-US-00002 TABLE 2 Stability results for JHU 2106 in different
matrices. Time Mouse Mouse Mouse Human Human (min) HBSS Plasma S9
Microsomes Plasma Microsomes 0 100% 100% 100% 100% 100% 100% 30 92%
102% 101% 102% 100% 90% 60 94% 101% 86% 103% 86% 96%
TABLE-US-00003 TABLE 3 Stability results for JHU 2108 in different
matrices. Time Mouse Mouse Mouse (min) HBSS Plasma S9 Microsomes 0
100% 100% 100% 100% 30 99% 2% 106% 99% 60 101% 1% 100% 100%
TABLE-US-00004 TABLE 4 Stability results for JHU 2109 in different
matrices. Time Mouse Mouse Mouse (min) HBSS Plasma S9 Microsomes 0
100% 100% 100% 100% 30 46% 0% 1% 2% 60 21% 0% 0% 0%
TABLE-US-00005 TABLE 5 Stability results for JHU 2110 in different
matrices. Time Mouse Mouse Mouse (min) HBSS Plasma S9 Microsomes 0
100% 100% 100% 100% 30 101% .sup. 2% .sup. 1% .sup. 2% 60 93% 0.4%
0.1% 0.1%
TABLE-US-00006 TABLE 6 Stability results for JHU 2111 in different
matrices. Time Mouse Mouse Mouse (min) HBSS Plasma S9 Microsomes 0
100% 100% 100% 100% 30 100% 3% 1% 3% 60 95% 1% 2% 2%
TABLE-US-00007 TABLE 7 Stability results for JHU 2112 in different
matrices. Time Mouse Mouse Mouse (min) HBSS Plasma S9 Microsomes 0
100% 100% 100% 100% 30 93% 2% .sup. 0% .sup. 2% 60 93% 1% 0.1%
0.1%
TABLE-US-00008 TABLE 8 Stability results for JHU 2236 in different
matrices. Time Mouse Mouse Mouse Human Human (min) Microsomes
Plasma S9 Microsomes plasma 0 100% 100% 100% 100% 100% 30 99% 99%
103% 88% 81% 60 88% 83% 103% 73% 59%
TABLE-US-00009 TABLE 9 Stability results for JHU 2237 in different
matrices. Time Mouse Mouse Mouse Human Human (min) Microsomes
Plasma S9 Microsomes plasma 0 100% 0% 100% 100% 100% 30 2% 0% 1% 1%
50% 60 0% 0% 0% 0% 18%
TABLE-US-00010 TABLE 10 Stability results for JHU 2608, 2609 and
2610 in different species Time Human Human Mouse Mouse JHU# (min)
Microsomes Plasma Microsomes Plasma JHU 0 100% 100% 100% 100% 2608
30 80% 109% 49% 0% 60 61% 76% 22% 0% JHU 0 100% 100% 100% 100% 2609
30 27% 106% 1% 0% 60 8% 75% 0% 0% JHU 0 100% 100% 100% 100% 2610 30
92% 98% 86% 3% 60 81% 92% 68% 1%
Example 12
2-PMPA Prodrugs with Tris POC Esters in Different Species
[0547] The 2-PMPA prodrugs with the Tris-POC esters showed
excellent oral bioavailability in rodents and dogs (compound JHU
2609, Table 10; compound JAM0186, FIGS. 11-14, Table 11). The
Tris-POC compound enhanced exposures following oral dosing in mice
and achieved more than 20-fold enhancement in permeability versus
2-PMPA (FIG. 12). The metabolic stability of the Tris-POC compound
in different species was seen, with the dog stability most similar
to the human stability (FIG. 13). Relative to the mouse, the dog
sample showed a 10 fold increase in the C.sub.max of 2-PMPA,
showing a high availability of the compound in the dog species
(FIG. 14). The metabolic stability of the Tris-POC compound could
be further enhanced in different species by the addition of a
methyl group (FIG. 15).
TABLE-US-00011 TABLE 11 Stability results for TRIS-POC in different
species Time Human Human Mouse Mouse (min) Microsomes Plasma
Microsomes Plasma 0 100% 100% 100% 100% 30 3% 64% 1% 1% 60 0% 48%
0% 0% Time Human Human Dog Dog Monkey Monkey (min) Microsomes
Plasma Microsomes Plasma Microsomes Plasma 0 100% 100% 100% 100%
100% 100% 30 5% 102% 1% 67% 0% 50% 60 0% 77% 0% 28% 0% 11%
Example 13
Pharmacological Inhibition of PSMA as IBD Therapy
[0548] An Overview of IBD:
[0549] IBD, an idiopathic, chronic and frequently disabling
inflammatory disorder of the intestine, has two subtypes: Crohn's
disease (CD) and ulcerative colitis (UC), each accounting for
.about.50% of IBD patients (Xavier and Podolsky, 2007; Strober et
al., 2007; Sartor, 2006). IBD is a widespread GI disease, with a
prevalence of -0.2% in Western population. In the United States
alone, there are 1.4 million diagnosed IBD patients, resulting in
enormous suffering and health-care costs. It is increasingly clear
that IBD is a complex multifactorial disease with both genetic and
environmental contributions, the interaction of which leads to IBD
(Xavier and Podolsky, 2007; Strober et al., 2007; Sartor, 2006;
Kaser et al., 2010). Unfortunately, the etiology of this mucosal
dysregulation in UC and CD remain elusive (Kaser et al., 2010).
Despite increasing therapeutic options available for the management
of IBD, approximately 1/3 of IBD patients do not respond to any
given therapy, and there is no cure for IBD (Hamilton et al.,
2012). Anti-tumor necrosis factor (TNF)-based therapies, such as
infliximab (IFX), adalimumab and certolizumab pegol are currently
the most effective therapies for severe UC and CD (Hanauer et al.,
2002; Kozuch and Hanauer, 2008; Colombel et al., 2007; Schreiber et
al., 2007). However, one-third of patients with CD do not respond
to anti-TNF therapies and another third lose responsiveness within
six months of initiating therapy (Regueiro et al., 2007; Lawrance,
2014). These nonresponders have more aggressive mucosal immune
responses and additional treatments are indicated (Schmidt et al.,
2007). Patients with extensive disease or who are at risk for short
gut syndrome due to prior resections are usually poor surgical
candidates. Currently, the only approved medication for patients
who have failed an anti-TNF agent is natalizumab. However,
natalizumab has been associated with several cases of progressive
and often fatal multifocal leukoencephalopathy (PML; Van et al.,
2005). This emphasizes the significance of exploring and
identifying new and more effective therapies in patients with
IBD.
[0550] Human Validation Data:
[0551] PSMA expression and enzymatic activity is selectively
elevated in patient samples with IBD (FIGS. 16-17). Gene-profiling
and immunohistological analyses (FIG. 16) showed that PSMA is
intensely upregulated in the intestinal mucosa of patients with
Crohn's disease (Zhang et al., 2012). To further determine the
relevance of PSMA to IBD, PSMA functional enzymatic activity was
examined and compared in normal and diseased mucosa of 32 surgical
intestinal specimens from 20 subjects (FIG. 17), including healthy
controls, patients with IBD, and non-IBD controls (diverticulitis),
using previously described methods. A 300-1,000% increase in PSMA
activity was found in the intestinal mucosa with active IBD when
compared to that in an uninvolved area of the same patients, or the
intestine from healthy and non-IBD controls. These data suggest a
clear positive association between activation of PSMA and IBD.
[0552] Preclinical Efficacy:
[0553] PSMA inhibition shows profound efficacy in three major
animal models of IBD (FIGS. 18-21). To investigate whether PSMA can
be a suitable novel therapeutic target for clinical intervention
against IBD, the effect of PSMA prototype inhibitors on three most
widely used murine models of IBD was tested, including DNBS-induced
colitis, DSS-induced colitis, and IL-10 knockout (IL-10 KO) mice (a
genetic model that develops spontaneous colitis). In all three
models, PSMA inhibitor treatment dramatically ameliorated symptoms.
In the DNBS-induced colitis model (FIG. 18), PSMA inhibition was
found to be similar to positive control sulfasalazine. In the DSS
colitis model, PSMA inhibition significantly reduced the disease
activity index (FIG. 19). Moreover, the PSMA activity in the
colonic and cecal mucosa of DSS-treated mice was potently inhibited
by PSMA inhibitor, indicating target engagement (FIG. 20). The
efficacy of 2-PMPA in treatment of spontaneous colitis in IL-10 KO
mice was also remarkable. First, PSMA inhibitor 2-PMPA
significantly reduced the disease severity, including macroscopic
disease, colonic hypotrophy, and provided better stool consistency
(FIGS. 21A-21B). More interestingly, a complete retraction of
prolapse in 2 of the 20 mice (10%) treated with the inhibitor was
observed (FIG. 21D), a phenomenon that has never been seen in more
than 800 IL-10 KO mice used. The improvement of these
prolapse-retracting mice was unequivocally obvious in that their
body weight increased dramatically when compared to that of
untreated control IL-10 KO mice (FIG. 21C). In conclusion, using
three major animal models of IBD, the significance of PSMA as a
novel therapeutic target for treatment of IBD has been
demonstrated.
[0554] Novel Orally Available Prodrug of 2-PMPA has been Identified
that Exhibits>20 Fold Enhancement in 2-PMPA Permeability In
Vivo:
[0555] The very potent phosphonic acid-based PSMA inhibitor termed
2-PMPA (Ki=300 pM) (Rais et al., 2014) demonstrated excellent
efficacy following i.p. administration at 100 mg/kg in both the DSS
as well as IL 10 knock out model. However, 2-PMPA is extremely
hydrophilic with poor oral availability (F<1%). Given the
success of using prodrug approaches to increase the oral
bioavailability of other phosphonic acid drugs (Hepsera.TM. and
Viread.TM.) (Barditch-Crovo et al., 1997; Cundy et al., 1997;
Barditch-Crovo et al., 2001), a similar strategy for 2-PMPA was
employed. An orally bioavailable prodrug of 2-PMPA
(Tris-POC-2-PMPA) has been identified that enables .about.20 fold
enhancement in permeability (FIG. 17). More importantly, the
prodrug afforded>10-20 fold sustained concentrations of
liberated 2-PMPA for up to 4 hours following oral
administration.
[0556] Dose Response/Efficacy and Pharmacokinetic Studies of PSMA
Inhibitors (2-PMPA and its Oral Prodrug) in Two Murine Models of
IBD: IL-10 Knockout (KO) and DSS-Induced Colitis:
[0557] The dose response of 2-PMPA (HEPES saline as vehicle) with
three doses, 1, 10, and 100 mg/kg, using an i.p. delivery route,
has been completed and it was observed that a dose dependent effect
with 100 mg/kg provided the most benefit. The 2-PMPA Tris POC
prodrug has also been tested in a preliminary experiment at one
high dose via the oral route (100 mg/kg) using a 50% PEG/water
vehicle. Unfortunately, the vehicle itself showed detrimental
effects. Several FDA approved vehicles will be evaluated including
ethanol/tween, propylene glycol and
2-Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) for solubility and
compatibility (Thackaberry, 2013). Once the optimal vehicle is
identified, a dose response of the oral 2-PMPA Tris POC prodrug
delivered at 3, 10, and 30 mg/kg p.o. (equivalent 2-PMPA) will be
completed. To evaluate efficacy, for DSS model of colitis, the
prodrug will be evaluated orally at three different doses mentioned
above at the same time (day 1) as DSS is given to induce colitis.
The treatment duration will be 7 days, as described in FIG. 23. For
IL-10 KO model of colitis, 3 month old mice will be given the
prodrug and treatment duration will be 2 weeks, as described in
FIG. 26.
[0558] For the pharmacokinetic studies, at the end of treatment on
day 7 (DSS model) and day 14 (IL-10 KO model) at 2 hour post dose,
blood and colonic mucosa will be collected for drug PK analysis.
Plasma will be generated from blood by centrifugation and all
samples will be stored at -80.degree. C. until further analysis.
Concentrations of inhibitors in plasma and tissue will be
determined via LC/MS/MS as described previously (Rais et al.,
2014).
[0559] Determine the Cellular and Molecular Mechanisms of PSMA
Inhibition in IBD Including Effects on Intestinal Epithelial Cells,
Dendritic Cells (DCs), and Intestinal Mucosal Cytokine
Profiles:
[0560] Involvement of PSMA in the pathogenesis of IBD is novel and
little is known. Explanation of this association is beyond the
current knowledge on PSMA. Therefore, it is important to understand
how PSMA is involved in IBD at the cellular and/or molecular
levels.
[0561] The major site of PSMA expression in the intestine is the
mucosa (FIGS. 21 and 22), where immunohistological analysis shows
PSMA is predominantly expressed in the intestinal epithelial cells
(FIG. 21). Therefore, to determine if the PSMA inhibitors target
directly to the intestinal epithelial cells, the first logical site
of drug action would be the intestinal epithelial cells. The
hypothesis is that in the state of IBD, intestinal epithelial
cells, which are the first defense line in the gut to keep the
commensal and invading bacteria at bay, may sense a change in
luminal bacteria and respond with a surge of PSMA expression. This
increase of PSMA activity would then promote subsequent secretion
of proinflammatory factors such as cytokines, and trigger an
inflammatory cascade that leads to IBD. To test this hypothesis,
colonic epithelial cells (CECs), both Caco-2 cells (a widely used
colonic epithelial cell line) and CECs isolated from mice (WT,
control and IL-10KO), will be used, as schematically illustrated in
FIG. 27. For normal CECs or Caco-2 cells, inflammatory conditions
will be induced by 10 nm LPS during cell culture (LPS will activate
the proinflammatory cascade through TLR4 that are highly expressed
on CECs). CECs isolated from IL-10KO mice (3-month old) will be
inflamed, and thus there is no need for induction with LPS.
Cytokine levels in culture medium (secreted) will be analyzed by
multiplex ELISA (using BIORAD Bio-Plex 200 System with HTF and
automated washer), as previously described (Alex et al., 2009; Alex
et al., 2010) for 17 different cytokines and chemokines, and those
in the cells will be analyzed by RT-PCR. The cytokines/chemokines
to be analyzed include IL-1.alpha., IL-1.beta., IL-2, IL-4, IL-6,
IL-10, IL-12 (p40), IL-13, IL-17, IFN (Interferon)-.gamma.,
TNF.alpha., G-CSF, CCL2 (MCP-1), CCL3 (MIP-1.alpha.), CCL4
(MIP-1.beta.), CXCL8 (IL-8), and CXCL10 (IP-10). It will be
determined if LPS treatment activates the gene expression of PSMA
by directly measuring PSMA activity. If PSMA inhibitors suppress
the expression and secretion of proinflammatory cytokines and/or
enhance the expression and secretion of anti-inflammatory cytokines
(such as IL-10 and/or IL-22), it would suggest that increased PSMA
expression promotes inflammation in CECs, and thereby confirms the
hypothesis that the PSMA inhibitors indeed target directly to the
CECs.
[0562] Another target for PSMA inhibitors might be the highly
specialized dendritic cells (DCs). IBD has been considered as a
T-cell-driven inflammatory disease, by highly specialized immune
cells called dendritic cells (DCs). DCs determine whether T-cell
responses are immunogenic (against harmful invading pathogens) or
tolerogenic (against harmless antigens). In the gut, intestinal DCs
recognize and respond to bacteria from the gut lumen and maintain
intestinal immune homeostasis by generating tolerogenic T-cell
responses towards the commensal microbiota. Previous efforts have
recently demonstrated a specific DC subset, CD103+ DCs, which can
be successfully identified in the human colon. The proportion of
CD103+ DCs was reduced in patients with active IBD (FIG. 28).
Recent reports in mice demonstrate intestinal DCs expressing the
gut-homing marker .alpha..sub.4.beta..sub.7 are required for
induction of T-reg and IL-10-producing T-cells (Villablanca et al.,
2013). There is strong evidence that expression of
.alpha..sub.4.beta..sub.7 on murine colonic DCs is confined to the
CD103+ subset. Furthermore, proportions of
.alpha..sub.4.beta..sub.7+ DCs are reduced in the inflamed colon of
IL-10KO mice (FIG. 29), suggesting IL-10 may play a key role in
differentiation of .alpha..sub.4.beta..sub.7+ regulatory DC subsets
in the gut. This subset of DCs plays a critical role of dampening
the T-cell response in normal condition and is lost in the
inflammatory condition in both human and murine model of
colitis.
[0563] Without wishing to be bound to any one particular theory, it
is thought that a PSMA inhibitor may up-regulate this particular
tolerogenic DC subset, thereby reducing the T-cell-mediated
inflammatory response and ameliorate symptoms of colitis in IL-10
KO mice as was observed (FIG. 26). Although PSMA is not normally
expressed in the DCs, it is possible that it is expressed in these
cells when under inflammatory conditions, and that PSMA expression
may suppress the expansion of this specific subset of DCs.
Alternatively, it is also possible that the upregulation of PSMA in
CECs activates and releases certain cellular factors that inhibit
the colonic DC expansion, resulting in the loss of this tolerogenic
DCs and leading to a hyper-reactive T-cell response. To test this
hypothesis, it will first be determined whether PSMA is upregulated
in DCs in the inflammatory condition. The best model for this
purpose is the IL-10KO mice, since previous efforts have already
demonstrated the therapeutic efficacy of PSMA inhibitors and the
loss of the colonic tolerogenic CD103+/.alpha..sub.4.beta..sub.7+
DC subset in IL-10KO mice. At least one of the following two
approaches can be employed: 1) RT-PCR: DCs can be isolated by FACS
from colonic mucosa of both WT (control) and IL-10KO mice. PSMA
expression can be analyzed by RT-PCR; 2) Immunohistology: Colonic
segments of both WT (control) and IL-10KO mice can be examined for
the expression of PSMA in DCs using CD103 and
.alpha..sub.4.beta..sub.7 as marker for DCs (triple labeling).
[0564] To determine if PSMA inhibitors promote tolerogenic
CD103+/.alpha..sub.4.beta..sub.7+ DC subset, colonic DCs can be
isolated from IL-10 KO mice that are treated or not (control) with
2-PMPA or its prodrug, and further analyzed for
CD103+/.alpha..sub.4.beta..sub.7+by FACS, as demonstrated in FIGS.
28-29. If the hypothesis is correct, it is expected that an
increase or recovery of the tolerogenic
CD103+/.alpha..sub.4.beta..sub.7+ DC subset toward what occurs in
WT mice (see FIG. 29) in the colon of diseased IL-10KO mice will be
seen.
[0565] In terms of cytokine profiling in the colonic mucosa, it is
hypothesized that inhibition of PSMA in general may suppress the
proinflammatory cytokines/chemokine and/or enhance
anti-inflammatory ones in the colon. To test this hypothesis, total
colonic mucosa can be isolated from colitis mice that are treated
or not (controls), and a set of 17 cytokines/chemokines (see the
list above) in the total mucosal protein extract can be analyzed by
multiplex ELISA.
[0566] Summary:
[0567] Recent genomic, clinical, and pharmacological data implicate
the metalloenzyme Prostate Specific Membrane Antigen (PSMA), in the
etiology of inflammatory bowel disease (IBD). Data illustrate that
pharmacological inhibition of PSMA using prototype inhibitors
ameliorates IBD symptoms in three preclinical models. Orally
available inhibitors have recently been synthesized and
characterized. Given these strong findings, it is hypothesized that
PSMA activates a proinflammatory signaling cascade that leads to or
enhances intestinal inflammation in IBD, and that specific
pharmacological inhibition will be a novel and effective strategy
for IBD therapy.
Example 14 (Prophetic)
Pharmacological Inhibition of PSMA as MS Therapy
[0568] Introduction:
[0569] Approximately 50% of 2.3 million Multiple Sclerosis (MS)
patients worldwide experience cognitive impairment, for which there
is no approved treatment (Dutta and Trapp. Neurology, 2007. 68(22
Suppl 3): p. S22-31; Calabrese, et al. Arch Neurol, 2009. 66(9): p.
1144-50), making therapies in MS cognition a large unmet medical
need. N-acetylaspartylglutamate (NAAG), one of the most abundant
neuropeptides in the mammalian brain (Neale, et al. J Neurochem,
2000. 75(2): p. 443-52), is thought to serve as the endogenous
agonist of the metabotropic glutamate receptor 3 (mGluR3)
(Olszewski, et al. Schizophr Res, 2012. 136(1-3): p. 160-1). Recent
clinical data collected in MS patients at Johns Hopkins University
revealed a significant positive correlation between hippocampal
NAAG concentration and patients' performances on a battery of
cognitive tasks (Rahn, et al. Proc Natl Acad Sci USA, 2012.
109(49): p. 20101-6). Notably, MS patients with low hippocampal
NAAG levels showed cognitive impairment while patients with higher
levels of hippocampal NAAG exhibited normal cognition. In support,
polymorphisms of mGluR3 have recently been linked to differential
cognitive abilities (Jablensky, et al. Genes Brain Behav, 2011.
10(4): p. 410-7; Egan, et al. Proc Natl Acad Sci USA, 2004.
101(34): p. 12604-9; 8Harrison, et al. J Psychopharmacol, 2008.
22(3): p. 308-22; Sartorius, et al. Neuropsychopharmacology, 2008.
33(11): p. 2626-34).
[0570] The brain metallopeptidase Glutamate Carboxypeptidase II
(GCPII) catabolizes NAAG in vivo. One of the most potent
(IC.sub.50=300 pM) and selective GCPII inhibitors, termed 2-PMPA,
has been shown to significantly increase brain NAAG levels and
improve cognition in preclinical models (Olszewski, et al., Transl
Psychiatry, 2012. 2: p. e145; Yamada, et al. Mol Pain, 2012. 8: p.
67; Janczura, et al., Eur J Pharmacol, 2013. 701(1-3): p. 27-32;
Gurkoff, et al. Brain Res, 2013. 1515: p. 98-107) including MS
(Rahn, et al. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6).
To our knowledge, 2-PMPA is the first and only treatment strategy
that has been shown to attenuate cognitive impairment in a
preclinical model of MS. However, 2-PMPA is a polar
bisphosphonate-based compound which is active only after systemic
dosing (i.p. or i.v.). It has negligible oral bioavailability and
is therefore unsuitable for daily chronic dosing in patients. Using
prodrug strategies proven successful in enhancing the oral
bioavailability of other bisphosphonate compounds which are now
marketed and widely used (ADEFOVIR.TM. and TENOFOVIR.TM.) (Cundy,
et al. J Pharm Sci, 1997. 86(12): p. 1334-8; Deeks, et al., J
Infect Dis, 1997. 176(6): p. 1517-23; Kearney, et al. Clin
Pharmacokinet, 2004. 43(9): p. 595-612), novel orally bioavailable
prodrugs of 2-PMPA can be synthesized. The presently disclosed
subject matter provides one such prodrug, with >100-fold
increase in bioavailability in dogs, respectively clearly
demonstrating feasibility of the approach. Employing an iterative
medicinal chemistry and drug metabolism/pharmacokinetic approach,
it is proposed to systematically optimize novel prodrugs with the
goal of developing an, ultimately, clinical investigation in MS
patients.
[0571] Prodrugs will be evaluated in an experimental autoimmune
encephalomyelitis (EAE) mouse model of multiple sclerosis. Mice
will be immunized and receive daily p.o. dosing of either vehicle
or 2-PMPA prodrug from the time of immunization until sacrifice
(prevention paradigm) or will be treated either with vehicle or
2-PMPA prodrug (treatment paradigm). The development and
progression of the resulting deficits will be tracked by EAE
disease scores, body weight measurements, and cognitive testing.
Post-mortem analysis of brain NAAG, 2-PMPA and GCPII inhibition
confirming target engagement will be performed in tandem.
[0572] NAAG is an mGluR3 Agonist which is Inactivated by GCPII:
[0573] N-acetyl aspartyl glutamate (NAAG), one of the most abundant
neuropeptides in the mammalian central nervous system (CNS) (Neale,
et al. J Neurochem, 2000. 75(2): p. 443-52), is a selective agonist
at metabotropic glutamate receptor 3 (mGluR3) (Olszewski, et al.
Schizophr Res, 2012. 136(1-3): p. 160-1). As with other
neurotransmitter/modulators, the concentration of extracellular
NAAG is tightly regulated. A 94 kD class II membrane bound zinc
metalloenzyme termed glutamate carboxypeptidase II (GCPII, also
called NAALADase or NAAG peptidase) degrades into N-acetylaspartate
(NAA) and glutamate (FIG. 26).
[0574] Decreased Brain NAAG Associated with Cognitive
Impairment:
[0575] Human studies spanning two decades report that CNS NAAG
concentrations are altered in neurological diseases with comorbid
cognitive impairment (Jaarsma, et al. J Neurol Sci, 1994. 127(2):
p. 230-3; Rowland, et al., GABA, and NAAG in schizophrenia.
Schizophr Bull, 2013. 39(5): p. 1096-104; Tsai, et al., CNS. Brain
Res, 1991. 556(1): p. 151-6), including MS (Rahn, et al. Proc Natl
Acad Sci USA, 2012. 109(49): p. 20101-6). Historically post-mortem
immunohistochemical or HPLC/MS techniques were required for
quantitation of brain NAAG levels, however with the recent
development of advanced neuroimaging techniques and increased MRI
magnet strength (.gtoreq.3T), in vivo imaging of NAAG is now
possible. Recent clinical data collected at the Johns Hopkins
hospital demonstrate a significant and selective positive
correlation between hippocampal NAAG concentration in MS patients
and their performance on a battery of cognitive tasks (Rahn, et al.
Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6). Specifically,
MS patients with cognitive impairment have low hippocampal NAAG
levels while MS patients with normal cognition have higher levels
of hippocampal NAAG (FIG. 27).
[0576] No Clinically Available GCPII Inhibitor to Date:
[0577] Unfortunately, to date no GCPII inhibitor has high potential
for clinical translation. Eisai, Inc (formerly Guilford
Pharmaceuticals) developed an orally bioavailable, thiol-based
GCPII inhibitor which completed 2 Phase 1 studies. Although the
inhibitor was well-tolerated in Phase 1 (REF) subsequent
immunological toxicities observed in GLP primate studies halted its
development. Importantly the toxicity was not due to the GCPII
mechanism, but rather due to the thiol moiety in the compound. As a
class, thiol drugs have a risk of inducing hypersensitivity
reactions (REF). Given the large unmet medical need for, second
generation non-thiol GCPII inhibitors devoid of this immunological
risk for advancement into clinical development can be synthesized.
Beyond thiol inhibitors, the most potent, selective, and efficacy
inhibitors of GCPII described are phosphonic acid based, however
they have minimal orally bioavailability.
[0578] 2-PMPA Increases Brain NAAG and Prevents Cognitive Deficits
in a Mouse Model:
[0579] The metabolite NAAG is broken down by the enzyme GCPII.
Without wishing to be bound to any one particular theory, it is
thought that administration of 2-phosponomethyl pentanedioic acid
(2-PMPA), a potent and selective inhibitor of GCPII, would reverse
cognitive impairment in an animal model of MS with known learning
and memory deficits (Ziehn, et al. EAE. Lab Invest, 2010. 90(5): p.
774-86). Mice (n=10) were immunized for EAE, injections of 2-PMPA
were administered daily from the time of disease induction, and
behavior tests were conducted after chronic physical signs of
disease were established. While no difference in physical severity
was observed, cognition was significantly improved in mice treated
with the GCPII inhibitor (EAE+2-PMPA) compared to vehicle-treated
controls (EAE+Vehicle) as measured by Barnes maze (a circular land
maze analogous to the Morris water maze that is used for animals
with physical disabilities) and fear conditioning tests (Rahn, et
al. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6). EAE+Vehicle
mice had higher Barnes maze path efficiency delta and significantly
decreased total latency delta as compared to Control+Vehicle
(P<0.05), indicating cognitive impairment. Conversely, the total
latency and path efficiency of EAE+2-PMPA mice did not differ from
Control+Vehicle mice. Furthermore, EAE+2-PMPA mice had
significantly improved (i.e. over 2-fold) path efficiency and total
latency as compared to EAE+Vehicle mice (FIG. 28A and FIG. 28B, P
<0.01 and P<0.05, respectively). Fear conditioning tests
demonstrated a significant difference between fear memory in
EAE+2-PMPA mice compared to EAE+Vehicle mice (P<0.05). Post
mortem analysis demonstrated a significant increase in brain NAAG
in EAE+2-PMPA mice versus EAE+Vehicle mice (FIG. 28C, P<0.05).
Taken together, these data demonstrate that GCPII inhibition
restores the cognitive and biological deficits resulting from
EAE.
[0580] Conduct Prodrug Efficacy Studies in a Mouse Model of
Multiple Sclerosis:
[0581] Prodrugs of 2-PMPA will be tested for in vivo efficacy.
Preclinical studies will be conducted using the research design in
which daily intraperitoneal injection of the GCPII inhibitor 2-PMPA
demonstrated significant beneficial effects on cognitive function
(Rahn, et al. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6).
Mice will be immunized for chronic EAE as previously described
(Rahn, et al. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6),
and daily oral dosing of prodrug or vehicle will begin from the
time of immunization and continue until sacrifice. Control groups
not immunized for EAE will be included in to determine if daily
2-PMPA prodrug administration improves learning and memory in
healthy non-EAE control mice. An EAE+2-PMPA i.p. control group will
be included to determine if oral prodrug treatment is more or less
efficacious versus daily intraperitoneal injections 2-PMPA. Animals
will be divided into five groups (n=15/group):
[0582] Group 1=Control+Vehicle
[0583] Group 2=Control+2-PMPA Prodrug
[0584] Group 3=EAE+Vehicle
[0585] Group 4=EAE+2-PMPA Prodrug
[0586] Group 5=EAE+2-PMPA (i.p.)
[0587] Mice will be monitored daily for signs of EAE. Approximately
two weeks after disease onset, mice will be subjected to elevated
plus maze testing, followed by Barnes maze testing, then fear
conditioning. Upon completion of the tests (approximately Day 50),
animals will be sacrificed and brains will be dissected. NAA, NAAG
and 2-PMPA levels will be measured in the hippocampus, cerebellum,
and frontal lobe via mass spectrometry. To measure prodrug
bioavailability and the effects of GCPII inhibition on brain NAAG,
five satellite animals will be sacrificed prior to behavior testing
(approximately 4 weeks post-immunization). The remaining 10 animals
will be sacrificed following the completion of all behavioral
tests. The above tests will be conducted for 2-PMPA prodrugs.
[0588] Expected Results:
[0589] The GCPII inhibitor prodrug is thought to be equally
efficacious at preventing cognitive impairment in EAE as compared
to daily intraperitoneal injections of 2-PMPA. EAE mice treated
with the 2-PMPA prodrug are expected to perform as well as Control
(i.e. non-EAE) mice on Barnes maze and fear conditioning tests.
Elevated plus maze performance, a measure of anxiety, is not
expected to differ between EAE and Control cohorts. It is expected
that the prodrug treatment will restore brain NAAG levels
equivalent to those observed in Control mice (Rahn, et al. Proc
Natl Acad Sci USA, 2012. 109(49): p. 20101-6). Previous work from
our laboratory and others has demonstrated that GCPII inhibition
has no effect on cognitive function in Control mice. Therefore,
Groups 1 and 2 are not expected to differ with regard to cognitive
function. It is possible, however, that the improved
bioavailability of the prodrug will cause cognitive enhancing
effects in normal mice.
[0590] Summary:
[0591] Despite the fact that over 200,000 MS patients suffer from
some type of cognitive impairment in the United States alone, no
therapies have been developed to treat MS-associated learning and
memory deficits. The failure of clinical trials designed to
translate pre-existing therapies for other neurological diseases
with comorbid cognitive impairment, such as memantine,
rivastigmine, and donepezil in Alzheimer's disease, to treatments
for cognitive impairment in MS suggest that alternative and
specific treatment pathways should be explored. To our knowledge
the present approach is the first to develop a drug treatment that
selectively targets an established biological deficit in
cognitively impaired MS patients (i.e. the reduction in brain
NAAG). Thus, completion of the presently disclosed studies could
lead to the first treatment specifically developed to treat
cognitive impairment in MS. NMSS thought that our NAAG/GCPII
efficacy data were sufficiently important to fund mechanistic
research (PI: Dr. Adam Kaplin) into the action of 2-PMPA utilizing
GCPII and mGluR3 KO mice, and pharmacological receptor antagonists.
While related to Dr. Kaplin's project, this independent project is
a logical and translationally-focused continuation of the funded
work, as 2-PMPA is not an effective long-term treatment strategy in
humans. The presently disclosed studies are required to develop a
compound that is safe and effective for human use.
[0592] In addition to a novel treatment for cognitive impairment,
these studies may also lead to the development of a human biomarker
with clinical and treatment applications. Recent advances in
magnetic resonance spectroscopy allow for the quantitation of CNS
NAAG levels in humans using 3T or 7T MRI scanners. NAAG, therefore,
can be used as a disease biomarker to measure changes in NAAG
levels in MS patients over time, identify the .about.1 million
patients who would benefit from our treatment strategy, and monitor
drug effects over time or may be susceptible to cognitive
impairment.
REFERENCES
[0593] All publications, patent applications, patents, and other
references mentioned in the specification are indicative of the
level of those skilled in the art to which the presently disclosed
subject matter pertains. All publications, patent applications,
patents, and other references are herein incorporated by reference
to the same extent as if each individual publication, patent
application, patent, and other reference was specifically and
individually indicated to be incorporated by reference. It will be
understood that, although a number of patent applications, patents,
and other references are referred to herein, such reference does
not constitute an admission that any of these documents forms part
of the common general knowledge in the art. In case of a conflict
between the specification and any of the incorporated references,
the specification (including any amendments thereof, which may be
based on an incorporated reference), shall control. Standard
art-accepted meanings of terms are used herein unless indicated
otherwise. Standard abbreviations for various terms are used
herein.
[0594] Although the foregoing subject matter has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be understood by those skilled in
the art that certain changes and modifications can be practiced
within the scope of the appended claims. [0595] Alex, P.; Zachos,
N.C.; Nguyen, T.; Gonzales, L.; Chen, T. E.; Conklin, L. S.;
Centola, M.; Li, X. Distinct cytokine patterns identified from
multiplex profiles of murine DSS and TNBS-induced colitis. Inflamm.
Bowel Dis. 2009, 15:341-352. [0596] Alex, P.; Ye, M.; Zachos, N.
Z.; Sipes, J.; Nguyen, T.; Suhodrev, M.; Gonzales, L.; Arora, Z.;
Zhang, T.; Centola, M.; Guggino, S. E.; Li, X. Clc-5 Knockout mice
exhibit novel immunomodulatory effects and are more susceptible to
dextran sulphate sodium induced colitis. J. Immunol. 2010,
184:3988-3996. [0597] Barditch-Crovo, P.; Deeks, S. G.; Collier,
A.; Safrin, S.; Coakley, D. F.; Miller, M.; Kearney, B. P.;
Coleman, R. L.; Limy, PAD.; Kahn, J. O.; McGowan, I.; Lietman, P.
S. Phase i/ii trial of the pharmacokinetics, safety, and
antiretroviral activity of tenofovir disoproxil fumarate in human
immunodeficiency virus-infected adults. Antimicrob. Agents
Chemother. 2001, 45:2733-2739. [0598] Barditch-Crovo, P.; Toole,
J.; Hendrix, C. W.; Cundy, K. C.; Ebeling, D.; Jaffe, H. S.;
Lietman, P. S. Anti-human immunodeficiency virus (HIV) activity,
safety, and pharmacokinetics of adefovir dipivoxil
(9-[2-(bis-pivaloyloxymethyl)-phosphonylmethoxyethyl]adenine) in
HIV-infected patients. J. Infect. Dis. 1997, 176:406-413. [0599]
Colombel, J. F.; Sandborn, W. J.; Rutgeerts, P.; Enns, R.; Hanauer,
S. B.; Panaccione, R.; Schreiber, S.; Byczkowski, D.; Li, J.; Kent,
J. D.; Pollack, P. F., Adalimumab for maintenance of clinical
response and remission in patients with Crohn's disease: the CHARM
trial. Gastroenterology 2007, 132:52-65. [0600] Cundy, K. C.; Sue,
I. L.; Visor, G. C.; Marshburn, J.; Nakamura, C.; Lee, W. A.; Shaw,
J. P. Oral formulations of adefovir dipivoxil: in vitro dissolution
and in vivo bioavailability in dogs. J. Pharm. Sci. 1997,
86:1334-1338. [0601] Hamilton, M. J.; Snapper, S. B.; Blumberg, R.
S., Update on biologic pathways in inflammatory bowel disease and
their therapeutic relevance. J. Gastroenterol. 2012, 47:1-8. [0602]
Hanauer, S. B.; Feagan, B. G.; Lichtenstein, G. R.; Mayer, L. F.;
Schreiber, S.; Colombel, J. F.; Rachmilewitz, D.; Wolf, D. C.;
Olson, A.; Bao, W.; Rutgeerts, P., Maintenance infliximab for
Crohn's disease: the ACCENT I randomised trial. Lancet 2002,
359:1541-1549. [0603] Kaser, A., Zeissig, S., Blumberg, R. S.,
Inflammatory bowel disease. Annu. Rev. Immunol. 2010, 28:573-621.
[0604] Kozuch, P. L. and Hanauer, S. B., Treatment of inflammatory
bowel disease: A review of medical therapy. World J. Gastroenterol.
2008, 14:354-377. [0605] Lawrance, I. C. What is left when
anti-tumour necrosis factor therapy in inflammatory bowel diseases
fails? World J. Gastroenterol. 2014, 20:1248-1258. [0606] Mesters,
J. R.; Barinka, C.; Li, W.; Tsukamoto, T.; Majer, P.; Slusher, B.
S.; Konvalinka, J.; Hilgenfeld, R., Structure of glutamate
carboxypeptidase II, a drug target in neuronal damage and prostate
cancer. EMBO J. 2006, 25:1375-1384. [0607] Rais, R.; Rojas, C.;
Wozniak, K.; Wu, Y.; Zhao, M.; Tsukamoto, T.; Rudek, M. A.;
Slusher, B. S., Bioanalytical method for evaluating the
pharmacokinetics of the GCP-II inhibitor 2-phosphonomethyl
pentanedioic acid (2-PMPA). J. Pharm. Biomed. Anal. 2014,
88:162-169. [0608] Regueiro, M.; Siemanowski, B.; Kip, K. E.;
Plevy, S., Infliximab dose intensification in Crohn's disease.
Inflamm. Bowel Dis. 2007, 13:1093-1099. [0609] Ristau, B. T.;
O'Keefe, D. S.; Bacich, D. J., The prostate-specific membrane
antigen: Lessons and current clinical implications from 20 years of
research. Urol. Oncol. 2013, 32(3):272-9. [0610] Sartor, R. B.,
Mechanisms of disease: pathogenesis of Crohn's disease and
ulcerative colitis. Nat. Clin. Pract. Gastroenterol. Hepatol. 2006,
3:390-407. [0611] Schreiber, S.; Khaliq-Kareemi, M.; Lawrance, I.
C.; Thomsen, O. O.; Hanauer, S. B.; McColm, J.; Bloomfield, R.;
Sandborn, W. J., Maintenance therapy with certolizumab pegol for
Crohn's disease. N Engl. J. Med. 2007, 357:239-250. [0612] Schmidt,
C.; Giese, T.; Hermann, E.; Zeuzem, S.; Meuer, S. C.; Stallmach,
A., Predictive value of mucosal TNF-alpha transcripts in
steroid-refractory Crohn's disease patients receiving intensive
immunosuppressive therapy. Inflamm. Bowel Dis. 2007, 13:65-70.
[0613] Slusher, B. S.; Rojas, C.; Coyle, J. T., Glutamate
Carboxypeptidase II. In: Rawlings and Salvesen, editors. Handbook
for Proteolytic Enzymes, Academic Press. 3rd Edition. 2013,
1620-1626. [0614] Strober, W.; Fuss, I.; and Mannon, P., The
fundamental basis of inflammatory bowel disease. J. Clin. Invest.
2007, 117:514-521. [0615] Thackaberry, E. A. Vehicle selection for
nonclinical oral safety studies. Expert Opin. Drug Metab. Toxicol.
2013, 9:1635-1646. [0616] Van, A. G.; Van, R. M.; Sciot, R.;
Dubois, B.; Vermeire, S.; Noman, M.; Verbeeck, J.; Geboe, s K.;
Robberecht, W.; Rutgeerts, P., Progressive multifocal
leukoencephalopathy after natalizumab therapy for Crohn's disease.
N. Engl. J. Med. 2005, 353:362-368. [0617] Villablanca, E. J.; De,
C. J.; Torregrosa, P. P.; Cassani, B.; Nguyen, D. D.; Gabrielsson,
S.; Mora, J. R. beta7 integrins are required to give rise to
intestinal mononuclear phagocytes with tolerogenic potential. Gut
2013, Sep. 12. [0618] Xavier, R. J. and Podolsky, D. K.,
Unravelling the pathogenesis of inflammatory bowel disease. Nature
2007, 448:427-434. [0619] Zhang, T.; Song, B.; Zhu, W.; Xu, X.;
Gong, Q. Q.; Morando, C.; Dassopoulos, T.; Newberry, R. D.; Hunt,
S. R.; Li, E., An ileal Crohn's disease gene signature based on
whole human genome expression profiles of disease unaffected ileal
mucosal biopsies. PLoS ONE 2012; 7:e37139. [0620] Dutta, R. and B.
D. Trapp, Pathogenesis of axonal and neuronal damage in multiple
sclerosis. Neurology, 2007. 68(22 Suppl 3): p. S22-31; discussion
S43-54. [0621] Calabrese, M., et al., Cortical lesions and atrophy
associated with cognitiveimpairment in relapsing-remitting multiple
sclerosis. Arch Neurol, 2009. 66(9): p. 1144-50. [0622] Neale, J.
H., T. Bzdega, and B. Wroblewska, N-Acetylaspartylglutamate: the
most abundant peptide neurotransmitter in the mammalian central
nervous system. J Neurochem, 2000. 75(2): p. 443-52. [0623]
Olszewski, R. T., T. Bzdega, and J. H. Neale, mGluR3 and not mGluR2
receptors mediate the efficacy of NAAG peptidase inhibitor in
validated model of schizophrenia. Schizophr Res, 2012. 136(1-3): p.
160-1. [0624] Rahn, K. A., et al., Inhibition of glutamate
carboxypeptidase II (GCPII) activity as a treatment for cognitive
impairment in multiple sclerosis. Proc Natl Acad Sci USA, 2012.
109(49): p. 20101-6. [0625] Jablensky, A., et al., Polymorphisms
associated with normal memory variation also affect memory
impairment in schizophrenia. Genes Brain Behav, 2011. 10(4): p.
410-7. [0626] Egan, M. F., et al., Variation in GRM3 affects
cognition, prefrontal glutamate, and risk for schizophrenia. Proc
Natl Acad Sci USA, 2004. 101(34): p. 12604-9. [0627] Harrison, P.
J., et al., The group II metabotropic glutamate receptor 3 (mGluR3,
mGlu3, GRM3): expression, function and involvement in
schizophrenia. J Psychopharmacol, 2008. 22(3): p. 308-22. [0628]
Sartorius, L. J., et al., Expression of a GRM3 splice variant is
increased in the dorsolateral prefrontal cortex of individuals
carrying a schizophrenia risk SNP. Neuropsychopharmacology, 2008.
33(11): p. 2626-34. [0629] Olszewski, R. T., et al., NAAG peptidase
inhibitors block cognitive deficit induced by MK-801 and motor
activation induced by d-amphetamine in animal models of
schizophrenia. Transl Psychiatry, 2012. 2: p. e145. [0630] Yamada,
T., et al., NAAG peptidase inhibition in the periaqueductal gray
and rostral ventromedial medulla reduces flinching in the formalin
model of inflammation. Mol Pain, 2012. 8: p. 67. [0631] Janczura,
K. J., et al., NAAG peptidase inhibitors and deletion of NAAG
peptidase gene enhance memory in novel object recognition test. Eur
J Pharmacol, 2013. 701(1-3): p. 27-32. [0632] Gurkoff, G. G., et
al., NAAG peptidase inhibitor improves motor function and reduces
cognitive dysfunction in a model of TBI with secondary hypoxia.
Brain Res, 2013. 1515: p. 98-107. [0633] Cundy, K. C., et al., Oral
formulations of adefovir dipivoxil: in vitro dissolution and in
vivo bioavailability in dogs. J Pharm Sci, 1997. 86(12): p. 1334-8.
[0634] Deeks, S. G., et al., The safety and efficacy of adefovir
dipivoxil, a novel anti-human immunodeficiency virus (HIV) therapy,
in HIV-infected adults: a randomized, double-blind,
placebo-controlled trial. J Infect Dis, 1997. 176(6): p. 1517-23.
[0635] Kearney, B. P., J. F. Flaherty, and J. Shah, Tenofovir
disoproxil fumarate: clinical pharmacology and pharmacokinetics.
Clin Pharmacokinet, 2004. 43(9): p. 595-612. [0636] Jaarsma, D., L.
Veenma-van der Duin, and J. Korf, N-acetylaspartate and
N-acetylaspartylglutamate levels in Alzheimer's disease post-mortem
brain tissue. J Neurol Sci, 1994. 127(2): p. 230-3. [0637] Rowland,
L. M., et al., In vivo measurements of glutamate, GABA, and NAAG in
schizophrenia. Schizophr Bull, 2013. 39(5): p. 1096-104. [0638]
Tsai, G. C., et al., Reductions in acidic amino acids and
N-acetylaspartylglutamate in amyotrophic lateral sclerosis CNS.
Brain Res, 1991. 556(1): p. 151-6. [0639] Ziehn, M. O., et al.,
Hippocampal CA1 atrophy and synaptic loss during experimental
autoimmune encephalomyelitis, EAE. Lab Invest, 2010. 90(5): p.
774-86. [0640] Jackson, P. F., et al., Design and pharmacological
activity of phosphinic acid based NAALADase inhibitors. J Med Chem,
2001. 44(24): p. 4170-5. [0641] Jackson, P. F., et al., Design,
synthesis, and biological activity of a potent inhibitor of the
neuropeptidase N-acetylated alpha-linked acidic dipeptidase. J Med
Chem, 1996. 39(2): p. 619-22. [0642] Slusher, B. S., et al.,
Selective inhibition of NAALADase, which converts NAAG to
glutamate, reduces ischemic brain injury. Nat Med, 1999. 5(12): p.
1396-402. [0643] Rais, R., et al., Bioanalytical method for
evaluating the pharmacokinetics of the GCP-II inhibitor
2-phosphonomethyl pentanedioic acid (2-PMPA). J Pharm Biomed Anal,
2013. 88: p. 162-9.
[0644] Although the foregoing subject matter has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be understood by those skilled in
the art that certain changes and modifications can be practiced
within the scope of the appended claims.
* * * * *